阅读说明：本技术 优化的结合gp41的分子及其用途 (Optimized gp 41-binding molecules and uses thereof ) 是由 郭家颖 君朵·迪瑞希 J·L·诺德斯特姆 刘丽勤 莱斯利·S·约翰逊 S·凯尼格 B·F 于 2019-05-13 设计创作，主要内容包括：本发明涉及具有降低的免疫原性的优化的结合HIV-1 gp41的分子。更具体地,本发明涉及包括结合gp41的可变轻链(VL)结构域和/或结合gp41的可变重链(VH)结构域的优化的结合gp41的分子,其已经被优化以在施用至接受受试者时降低这种结构域的免疫原性。本发明特别涉及为多特异性结合gp41的分子(包括双特异性双抗体(包括双抗体)、双特异性抗体、三价结合分子(包括TRIDENT～(TM)分子)等)的结合gp41的分子,其包括：(i)这种优化的结合gp41的可变结构域和(ii)能够结合存在于效应细胞的表面上的分子的表位的结构域。本发明还涉及包括任何这种结合gp41的分子的药物组合物,和涉及任何这种结合gp41的分子在治疗HIV-1感染中的用途的方法。(The present invention relates to optimized molecules that bind to HIV-1gp41 with reduced immunogenicity. More specifically, the invention relates to optimized gp 41-binding molecules comprising a Variable Light (VL) domain that binds gp41 and/or a Variable Heavy (VH) domain that binds gp41, which have been optimized to reduce the immunogenicity of such domains when administered to a recipient subject. The invention particularly relates to molecules (including bispecific diabodies) (including Double antibodies), Bispecific antibodies, trivalent binding molecules (including TRIDENT) TM Molecule) etc.) comprising: (i) this optimized binding to the variable domain of gp41 and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions comprising any such gp 41-binding molecule, and to methods of using any such gp 41-binding molecule in the treatment of HIV-1 infection.)

1. A gp 41-binding molecule comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:57 and/or the VH domain comprises the amino acid sequence of SEQ ID NO: 58.

2. The gp 41-binding molecule of claim 1, wherein the gp 41-binding molecule comprises:

(a) the VL domain comprising the amino acid sequence of SEQ ID NO 57; and

(b) 58 comprising the amino acid sequence of SEQ ID NO.

3. The molecule that binds gp41 according to any one of claims 1-2, wherein the molecule is an antibody or includes a portion thereof that binds to an epitope of gp 41.

4. The molecule that binds gp41 according to any one of claims 1-3, wherein said molecule is:

(a) a bispecific antibody; or

(b) A diabody that is a covalently bound complex comprising two, three, four, or five polypeptide chains; or

(c) A trivalent binding molecule that is a covalently bound complex comprising three, four, five, or more than five polypeptide chains.

5. The molecule that binds gp41 according to claim 4, wherein the molecule is the diabody and comprises an albumin-binding domain (ABD).

6. The molecule that binds gp41 according to any one of claims 1-4, wherein said molecule comprises an Fc region.

7. The gp 41-binding molecule of claim 5, wherein the Fc region is a variant Fc region comprising:

(a) one or more amino acid modifications that reduce the affinity of the variant Fc region for fcyr; and/or

(b) One or more amino acid modifications that enhance the serum half-life of the variant Fc region.

8. The molecule that binds gp41 of claim 7, wherein said modification that reduces the affinity of the variant Fc region for FcyR comprises L234A; L235A; or a substitution of L234A and L235A, wherein the numbering is that of the EU index as in Kabat.

9. The gp 41-binding molecule of any one of claims 7 or 8, wherein the modification that enhances the serum half-life of the variant Fc region comprises M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, wherein the numbering is that of the EU index as in Kabat.

10. The molecule that binds gp41 according to any one of claims 1-9, wherein the molecule is bispecific and comprises a site capable of immunospecifically binding to one binding epitope of an epitope of gp41 and a site capable of immunospecifically binding to one binding epitope of an epitope of a molecule present on the surface of an effector cell.

11. The molecule that binds gp41 according to any one of claims 1-9, wherein the molecule is bispecific and comprises two binding epitope sites capable of immunospecifically binding to an epitope of gp41 and two binding epitope sites capable of immunospecifically binding to an epitope of a molecule present on the surface of an effector cell.

12. The molecule that binds gp41 according to any one of claims 1-9, wherein the molecule is trispecific and comprises:

(a) a binding epitope site capable of immunospecifically binding to an epitope of gp 41;

(b) a binding epitope site capable of immunospecifically binding to an epitope of a first molecule present on the surface of an effector cell; and

(c) a site that binds an epitope that is capable of immunospecifically binding to an epitope of a second molecule present on the surface of an effector cell.

13. The molecule that binds gp41 according to any one of claims 1-9, wherein the molecule is trispecific and comprises:

(a) a binding epitope site capable of immunospecifically binding to a first epitope of gp 41;

(b) a site capable of immunospecifically binding to a second epitope of gp41 or to a binding epitope of a different epitope of the HIV-1 protein; and

(c) an epitope-binding site capable of immunospecifically binding to an epitope of a molecule present on the surface of an effector cell;

wherein the first epitope and the second epitope of gp41 are different.

14. The molecule that binds gp41 according to any one of claims 10-13, wherein the molecule is capable of simultaneously binding gp41 and the molecule present on the surface of an effector cell.

15. The molecule that binds gp41 according to any one of claims 10-14, wherein the molecule present on the surface of an effector cell is CD2, CD3, CD8, CD16, TCR, NKp46 or NKG 2D.

16. The molecule that binds gp41 according to any one of claims 10-15, wherein the effector cell is a cytotoxic T cell or a Natural Killer (NK) cell.

17. The molecule that binds gp41 according to claim 15, wherein the molecule present on the surface of an effector cell is CD 3.

18. The molecule that binds gp41 according to claim 12, wherein the first molecule present on the surface of an effector cell is CD3 and the second molecule present on the surface of an effector cell is CD 8.

19. The molecule that binds gp41 according to any one of claims 10-18, wherein said molecule mediates coordinated binding of gp 41-expressing cells and cytotoxic T cells.

20. The molecule that binds gp41 according to any one of claims 1-4 and 6-9, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, and wherein:

I) (ii) (a) said first polypeptide chain comprises SEQ ID NO: 113;

(b) the second polypeptide chain comprises SEQ ID NO 114; and is

(c) The third polypeptide chain comprises SEQ ID NO 112;

or

II) (a) said first polypeptide chain comprises SEQ ID NO 115;

(b) the second polypeptide chain comprises SEQ ID NO: 116; and is

(c) The third polypeptide chain comprises SEQ ID NO 112.

21. The molecule that binds gp41 according to any one of claims 1-4 and 6-9, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, and wherein:

(a) the first polypeptide chain comprises SEQ ID NO 113;

(b) the second polypeptide chain comprises SEQ ID NO 114;

(c) the third polypeptide chain comprises SEQ ID NO: 117; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 118.

22. The molecule that binds gp41 according to any one of claims 1-4 and 6-9, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, and wherein:

I) (ii) (a) said first polypeptide chain comprises SEQ ID NO: 113;

(b) the second polypeptide chain comprises SEQ ID NO 114;

(c) the third polypeptide chain comprises SEQ ID NO 119; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 120;

or

II) (a) said first polypeptide chain comprises SEQ ID NO 115;

(b) the second polypeptide chain comprises SEQ ID NO: 116;

(c) the third polypeptide chain comprises SEQ ID NO 119; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 120.

23. A pharmaceutical composition comprising an effective amount of a molecule that binds gp41 according to any one of claims 1-22 and a pharmaceutically acceptable carrier.

24. A method of treating or preventing HIV-1 infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a molecule that binds gp41 according to any one of claims 1-22 or a pharmaceutical composition according to claim 23.

25. The method of claim 24, wherein the method further comprises administering a latent activator.

26. The method of claim 25, wherein the latency activator is vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryoid, PMA, ionomycin, or any combination thereof.

Technical Field

The present invention relates to optimized molecules that bind to HIV-1gp41 with reduced immunogenicity. More specifically, the invention relates to optimized gp 41-binding molecules comprising a Variable Light (VL) domain that binds gp41 and/or a Variable Heavy (VH) domain that binds gp41, which have been optimized to reduce the immunogenicity of such domains when administered to a recipient subject. The invention particularly relates to molecules (including bispecific diabodies (includingDouble antibodies),Bispecific antibodies, trivalent binding molecules (including TRIDENT)^TMMolecule) etc.) comprising: (i) this optimized binding to the variable domain of gp41 and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions comprising any such gp 41-binding molecule, and to methods of using any such gp 41-binding molecule in the treatment of HIV-1 infection.

Background

Highly active antiretroviral therapy (HAART) has been effective in reducing viral load and ameliorating the effects of HIV type 1(HIV-1) infection in infected individuals. However, HAART does not eradicate HIV-1 infection and it does not accelerate the elimination of infected cells. In infected individuals, HIV-1 persists in a latent state as a provirus integrated in resting memory CD4+ T cells, making HAART inaccessible. Thus, despite the administration of this therapy, the HIV-1 virus persists in individuals within the HIV-1 infected cell latency pool that have escaped treatment. CD8+ T cells have limited ability to eliminate HIV-1 from such latently infected cells.

Thus, there is a need for therapeutic agents for treating HIV-1 infected individuals, particularly agents that target virally infected cells and have the potential to reduce the latent pool of HIV-1 infected cells, as well as being suitable for repeated administration.

The envelope glycoprotein ("Env") of the HIV-1 virus is crucial for virus infectivity (via its ability to bind the CD4 receptor) and for driving membrane fusion. The HIV-1Env gene product consists of a trimeric complex of two subunits, gp120 and gp 41. The Env protein is synthesized as a glycosylated gp160 precursor protein that is folded into a trimer and proteolytically cleaved to yield the mature gp120 and gp41 proteins. Cleaved Env assembles with other viral components for virion budding From The Cell Surface and Is present on The Surface Of infected cells (Miranda, L., et al, 2002, "Cell Surface Expression Of The HIV-1Envelope Glycotens Is Directed From Intracellular CTLA-4-contained Regulated recombinant plasmids," Proc Natl Acad Sci U S A.99 (12): 8031-8036). Thus, HIV Env, and in particular its gp41 subunit, is a highly specific viral target for therapeutic elimination of a persistent pool of HIV infection via antibody-mediated cell killing (Sloane, D., et al, 2015, "Targeting HIV Reservoir in Infected CD 4T Cells by Dual-Affinity Re-Targeting Molecules (DARTs) that is close to HIV enveloppe and Recircuit Cytoxic T Cells" PLOS Pathologens | DOI:10.1371/j ournal. p.5233.

However, despite all previous advances, there is still a need for optimized gp 41-binding molecules with enhanced anti-HIV activity and/or reduced immunogenicity. The present invention addresses this need as well as the need for improved therapies for the treatment and prevention of HIV-1.

Disclosure of Invention

The present invention relates to optimized molecules that bind to HIV-1gp41 with reduced immunogenicity. More specifically, the invention relates to optimized gp 41-binding molecules comprising a Variable Light (VL) domain that binds gp41 and/or a Variable Heavy (VH) domain that binds gp41, which have been optimized to reduce the immunogenicity of such domains when administered to a recipient subject. The invention particularly relates to molecules (including bispecific diabodies) that bind gp41 as multispecific agentsDouble antibodies),Bispecific antibodies, trivalent binding molecules (including TRIDENT)^TMMolecule) etc.) comprising: (i) this optimized binding to the variable domain of gp41 and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell. The invention also relates to pharmaceutical compositions comprising any such gp 41-binding molecule, and to methods of using any such gp 41-binding molecule in the treatment of HIV-1 infection.

In detail, the present invention provides gp 41-binding molecules comprising a variable light chain (VL) domain and a variable heavy chain (VH) domain, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:57 and/or the VH domain comprises the amino acid sequence of SEQ ID NO: 58.

The present invention further relates to embodiments of the gp 41-binding molecule indicated above, wherein the gp 41-binding molecule comprises:

(a) a VL domain comprising the amino acid sequence of SEQ ID NO 57; and

(b) a VH domain comprising the amino acid sequence of SEQ ID NO: 58.

The invention further relates to embodiments of the above indicated molecules binding to gp41, wherein the molecule is an antibody or comprises a portion thereof binding to an epitope of gp 41.

The present invention further relates to embodiments of the above indicated gp 41-binding molecule, wherein the molecule is:

(a) a bispecific antibody; or

(b) Diabodies, which are covalently bound complexes comprising two, three, four, or five polypeptide chains; or

(c) A trivalent binding molecule that is a covalently bound complex comprising three, four, five, or more than five polypeptide chains.

The invention further relates to embodiments of the above indicated molecule that binds gp41, wherein the molecule is a diabody and comprises an Albumin Binding Domain (ABD).

The invention further relates to embodiments of the above-indicated gp 41-binding molecule, wherein the molecule comprises an Fc region. The invention further relates to embodiments of such molecules that bind gp41, wherein the Fc region is a variant Fc region comprising:

(a) one or more amino acid modifications that reduce the affinity of the variant Fc region for fcyr; and/or

(b) One or more amino acid modifications that enhance the serum half-life of the variant Fc region.

The invention further relates to embodiments of such molecules that bind gp41, wherein the modifications that reduce the affinity of the variant Fc region for fcyr include L234A; L235A; or a substitution of L234A and L235A, wherein the numbering is that of the EU index as in Kabat.

The invention further relates to embodiments of such molecules that bind gp41, wherein the modification that enhances the serum half-life of the variant Fc region comprises M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or a substitution of K288D and H435K, wherein the numbering is that of the EU index as in Kabat.

The invention further relates to embodiments of the above indicated molecules that bind gp41, wherein the molecule is bispecific and comprises a site capable of immunospecifically binding to one binding epitope of an epitope of gp41 and a site capable of immunospecifically binding to one binding epitope of an epitope of a molecule present on the surface of an effector cell.

The invention further relates to embodiments of the above indicated molecules binding to gp41, wherein the molecule is bispecific and comprises two binding epitope sites capable of immunospecifically binding to an epitope of gp41 and two binding epitope sites capable of immunospecifically binding to an epitope of a molecule present on the surface of an effector cell.

The present invention further relates to embodiments of the above-indicated molecule that binds gp41, wherein the molecule is trispecific and comprises:

(a) a binding epitope site capable of immunospecifically binding to an epitope of gp 41;

(b) a binding epitope site capable of immunospecifically binding to an epitope of a first molecule present on the surface of an effector cell; and

(c) a site that binds an epitope that is capable of immunospecifically binding to an epitope of a second molecule present on the surface of an effector cell.

The invention further relates to embodiments of such molecules that bind gp41, wherein the first molecule present on the surface of an effector cell is CD3 and the second molecule present on the surface of an effector cell is CD 8.

The present invention further relates to embodiments of the above-indicated molecule that binds gp41, wherein the molecule is trispecific and comprises:

(a) a binding epitope site capable of immunospecifically binding to a first epitope of gp 41;

(b) a site capable of immunospecifically binding to a second epitope of gp41 or to a binding epitope of a different epitope of the HIV-1 protein; and

(c) an epitope-binding site capable of immunospecifically binding to an epitope of a molecule present on the surface of an effector cell;

wherein the first epitope and the second epitope of gp41 are different.

The invention further relates to embodiments of the above indicated molecules binding to gp41, wherein the molecule is capable of simultaneously binding to gp41 and a molecule present on the surface of an effector cell. The invention further relates to embodiments of such molecules that bind gp41, wherein the molecule present on the surface of an effector cell is CD2, CD3, CD8, CD16, TCR, NKp46, or NKG 2D.

The invention further relates to embodiments of the above-indicated molecules that bind gp41, wherein the effector cell is a cytotoxic T cell or a Natural Killer (NK) cell.

The invention further relates to embodiments of the above-indicated molecules that bind gp41, wherein the molecules mediate coordinated binding of gp 41-expressing cells and cytotoxic T cells.

The invention further relates to embodiments of such a molecule that binds gp41, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, and wherein:

I) (a) the first polypeptide chain comprises SEQ ID NO: 113;

b) the second polypeptide chain comprises SEQ ID NO 114; and is

(c) The third polypeptide chain comprises SEQ ID NO 112;

or

II) (a) the first polypeptide chain comprises SEQ ID NO 115;

(b) the second polypeptide chain comprises SEQ ID NO 116; and is

(c) The third polypeptide chain comprises SEQ ID NO 112.

The invention further relates to embodiments of such a molecule that binds gp41, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain and wherein:

(a) the first polypeptide chain comprises SEQ ID NO 113;

(b) the second polypeptide chain comprises SEQ ID NO 114;

(c) the third polypeptide chain comprises SEQ ID NO 117; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 118.

The invention further relates to embodiments of such a molecule that binds gp41, wherein the molecule comprises a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain and wherein:

I) (a) the first polypeptide chain comprises SEQ ID NO: 113;

(b) the second polypeptide chain comprises SEQ ID NO 114;

(c) the third polypeptide chain comprises SEQ ID NO 119; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 120;

or

II) (a) the first polypeptide chain comprises SEQ ID NO 115;

(b) the second polypeptide chain comprises SEQ ID NO 116;

(c) the third polypeptide chain comprises SEQ ID NO 119; and is

(d) The fourth polypeptide chain comprises SEQ ID NO 120.

The invention further relates to a pharmaceutical composition comprising an effective amount of a gp 41-binding molecule of any one of claims 1-22 and a pharmaceutically acceptable carrier.

The present invention further relates to a method of treating or preventing HIV-1 infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising any one of the above-indicated molecules that binds gp41 or the above-indicated pharmaceutical composition.

The invention further relates to embodiments of such methods of treating or preventing HIV-1 infection, further comprising administering a latent activator (such as vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryoid, PMA, enomycin, or any combination thereof).

Brief Description of Drawings

FIGS. 1A-1B provide a schematic representation of a representative covalently bound diabody having two epitope binding domains composed of two polypeptide chains each having an E-helix or K-helix heterodimer promotion domain (alternate heterodimer promotion domains are provided below). Cysteine residues may be present in the linker (fig. 1A) and/or in the heterodimer promoting domain (fig. 1B). VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. The wavy line (WWW) in this figure and in all figures providing a schematic representation of the binding molecule domain represents one or more optional heterodimer promoting domains, which are preferably present.

Figure 2 provides a schematic representation of a representative covalently bound diabody molecule having two epitope binding domains consisting of two polypeptide chains, each polypeptide chain having a CH2 domain and a CH3 domain, such that the associated chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern.

Fig. 3A-3C provide schematic diagrams showing representative covalently bound tetravalent diabodies having four epitope binding domains composed of two pairs of polypeptide chains (i.e., a total of four polypeptide chains). One polypeptide chain in each pair is provided with a CH2 domain and a CH3 domain such that the associated chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. The two pairs of polypeptide chains can be identical. In such embodiments, where the two pairs of polypeptide chains are the same and the VL and VH domains recognize different epitopes (as shown in fig. 3A-3B), the resulting molecule possesses four epitope binding domains and is bispecific and bivalent for each bound epitope. In such embodiments, where the VL domain and VH domain recognize the same epitope (e.g., using the same VL domain CDRs and the same VH domain CDRs on both chains), the resulting molecule possesses four epitope binding domains and is monospecific and tetravalent with respect to a single epitope. Alternatively, the two pairs of polypeptides may be different. In such an embodiment, where the two pairs of polypeptide chains are different and the VL and VH domains of each pair recognize different epitopes (as shown by the different shading and patterning in fig. 3C), the resulting molecule possesses four epitope binding domains and is tetraspecific and monovalent for each binding epitope. Figure 3A shows diabodies containing an Fc region that contains a peptide heterodimer promoting domain that includes a cysteine residue. Figure 3B shows a diabody containing an Fc region that contains an E-helix and K-helix heterodimer-promoting domain including a cysteine residue and a linker (with an optional cysteine residue). Figure 3C shows a diabody containing an Fc region, which contains the antibody CH1 domain and the CL domain. As provided below, the VL/VH binding sites formed by association of polypeptide chains can be the same or different so as to allow tetravalent binding as monospecific, bispecific, trispecific, or tetraspecific.

FIGS. 4A-4B provide schematic diagrams of representative covalently bound diabody molecules having two epitope-binding domains composed of three polypeptide chains. Two of the polypeptide chains are provided with a CH2 domain and a CH3 domain such that the associated chains form all or part of an Fc region. The polypeptide chain comprising the VL domain and the VH domain further comprises a heterodimer facilitating domain. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern.

FIG. 5 provides a schematic of a representative covalently bound binding molecule having four epitope-binding domains consisting of five polypeptide chains. Two of the polypeptide chains are provided with a CH2 domain and a CH3 domain such that the associated chains form an Fc region that includes all or part of the Fc region. The polypeptide chain comprising the linked VL and VH domains further comprises a heterodimerization facilitating domain. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. As provided below, the VL/VH binding sites formed by association of polypeptide chains can be the same or different, so as to allow tetravalent binding to be monospecific, bispecific, trispecific, or tetraspecific.

FIGS. 6A-6F provide schematic representations of representative trivalent Fc region-containing binding molecules with three epitope binding domains. Fig. 6A schematically illustrates the domains of a trivalent binding molecule comprising two diabody-type binding domains and a Fab-type binding domain with different domain orientations, wherein the diabody-type binding domains are N-terminal to the Fc region. Fig. 6B schematically illustrates the domains of a trivalent binding molecule comprising two diabody-type binding domains and a Fab-type binding domain with different domain orientations, wherein the diabody-type binding domains are C-terminal to an Fc region. The molecules in fig. 6A-6B include four chains. Fig. 6C and 6D schematically illustrate the domains of a trivalent binding molecule comprising two diabody-type binding domains N-terminal to an Fc region and a Fab-type binding domain or an scFv-type binding domain wherein the light chain and the heavy chain are linked via a polypeptide spacer, respectively. The trivalent binding molecules in fig. 6E and 6F schematically illustrate the domains of a trivalent binding molecule comprising two diabody-type binding domains C-terminal to an Fc region and a Fab-type binding domain or scFv-type binding domain wherein the light and heavy chains are linked via a polypeptide spacer, respectively. The trivalent binding molecules in FIGS. 6C-6F include three chains. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern.

Fig. 7A-7B show an alignment of amino acid sequences of 7B2, germlined 7B2(7B2GL), and the indicated human germline. Differences from 7B2 are shaded, Frame (FW) and CDR regions are indicated and Kabat numbering is provided. The VL domain is presented in fig. 7A and the VL domain is presented in fig. 7B.

FIGS. 8A-8B show sensorgrams of recombinant HIV-1JRFL gp140(100, 50, 25nM) bound to immobilized 7B2 IgG (FIG. 8A) and 7B2GL IgG (FIG. 8B), as measured by Attana Cell A200 QCM.

Figure 9 shows the ability of DART-a (comprising the 7B2GL VL domain and VH domain optimized to minimize immunogenicity) and DART-1 (comprising the parent 7B2VL domain and VH domain) to bind to the surface of gp 140-mutexpressing HEK293/D371 cells.

Figure 10 shows the ability of DART-a (comprising the 7BGL VL domain and VH domain optimized to minimize immunogenicity) and DART-1 (comprising the parental 7B2VL domain and VH domain) to activate T cells as measured in the Jurkat T cell reporter assay.

Figure 11 shows the ability of DART-a (comprising the 7BGL VL domain and VH domain optimized to minimize immunogenicity) and DART-1 (comprising the parental 7B2VL domain and VH domain) to mediate T-cell redirected killing of target cells mutexpressing HIV gp41 in CTL assays.

Detailed Description

The present invention relates to optimized molecules that bind to HIV-1gp41 with reduced immunogenicity. More specifically, the invention relates to optimized gp 41-binding molecules comprising a variable light chain (VL) domain that has been optimized to bind gp41 and/or a variable heavy chain (VH) domain that binds gp41 to reduce the immunogenicity of such domains when administered to a recipient subject. The invention particularly relates to molecules (including bispecific diabodies) (includingDouble antibodies),Bispecific antibodies, trivalent binding molecules (including TRIDENT)^TMMolecule) etc.) comprising: (i) this optimized variable domain binding to gp41 and (ii) being able to bind to a molecule present on the surface of an effector cellThe domain of an epitope of (a). The invention also relates to pharmaceutical compositions comprising any such gp 41-binding molecule, and to methods of using any such gp 41-binding molecule in the treatment of HIV-1 infection.

I. Antibodies and other binding molecules

A. Antibodies

Molecules of the invention that bind gp41 can be an antibody, or can be derived from an antibody that binds gp41 (e.g., by fragmentation, cleavage, etc., of an antibody polypeptide), or obtained from the use of the amino acid sequence of one or more polypeptide chains of an antibody molecule, or expressed by, or obtained from the nucleotide sequence of, a polynucleotide encoding such a polypeptide.

Antibodies are immunoglobulin molecules that are capable of specifically binding to a particular domain or portion (motif) or construct ("epitope") of a molecule, such as a carbohydrate, polynucleotide, lipid, polypeptide, or the like. The epitope-containing molecule can have immunogenic activity such that it elicits an antibody-producing response in an animal; such molecules are referred to as "antigens". As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies (camelized antibodies), single chain fvs (scfv), single chain antibodies, Fab portions, F (ab') portions, disulfide-linked bispecific fvs (sdfv), intrabodies (intrabodies), and epitope-binding domains of any of the above. Such immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY); class (e.g., IgG)₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂) Or subclass; or species (bovine, equine, feline, canine, rodent, primate (e.g., including monkeys such as cynomolgus monkeys, humans, etc.)).

The term "monoclonal antibody" refers to a homogeneous population of antibodies, wherein a monoclonal antibody comprises amino acids (naturally occurring or non-naturally occurring) involved in selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term "monoclonal antibodyThe antibody "encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also portions thereof (e.g., Fab ', F (ab')₂(Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site that binds the desired specificity and ability of the antigen. The source of the antibody or the manner in which it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, etc.) is not intended to be limiting. The term includes intact immunoglobulins as well as portions thereof described above in accordance with the definition of "antibody" and the like.

Methods for making monoclonal antibodies are known in the art. One method that may be used is the method Of Kohler, G.et al (1975) "Continuous Cultures Of Fused Cells calibrating organism Of Predefined specification," Nature 256:495-497, or a modification thereof. Typically, monoclonal antibodies are developed in mice, rats or rabbits. Antibodies are produced by immunizing an animal with an immunogenic amount of cells, cell extracts, or protein preparations comprising the desired epitope. The immunogen may be, but is not limited to, a primary cell, a cultured cell line, a cancer cell, a protein, a peptide, a nucleic acid, or a tissue. The cells used for immunization can be cultured for a period of time (e.g., at least 24 hours) before they are used as immunogens. Cells may be Used as immunogens by themselves or in combination with non-denaturing Adjuvants such as Ribi (see, e.g., Jennings, V.M (1995) "Review of Selected Adjuvants Used in Antibody Production," ILAR J37 (3): 119-125). In general, when used as an immunogen, the cells should remain intact and preferably viable. Intact cells may allow the antigen to be better detected by the immunized animal than ruptured cells. The use of denaturing or harsh adjuvants (hash adjuvant), for example, freund's adjuvant, can rupture the cells and thus arrest them. The immunogen may be administered multiple times, such as biweekly or weekly, at periodic intervals, or may be administered in such a way as to maintain viability in the animal (e.g., in tissue recombinants). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the desired pathogenic epitope may be sequenced and recombinantly produced by any means known in the art. In one embodiment, such antibodies are sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequences encoding the antibody of interest may be maintained in a vector within the host cell, and the host cell may then be expanded and frozen for future use. The polynucleotide sequences of such antibodies can be used for genetic manipulation to produce monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules of the invention, as well as affinity-optimized chimeric, humanized, and/or caninized antibodies of the invention to improve the affinity or other characteristics of the antibodies, as described in detail below.

The antibodies and binding molecules of the invention bind epitopes via their binding domains in an "immunospecific" manner. As used herein, a molecule is considered to bind an epitope of another molecule in an immunospecific manner (or "immunospecifically") if the molecule binds or associates more frequently, more rapidly, for a longer duration, and/or with greater affinity to the epitope than the alternative epitope. For example, an antibody that immunospecifically binds to a viral epitope is an antibody that binds the viral epitope with greater affinity, avidity, more readily, and/or for a longer duration than it immunospecifically binds to other viral epitopes or non-viral epitopes. It will also be understood by reading this definition that, for example, an antibody (or portion or epitope) that immunospecifically binds to a first target may specifically or preferentially bind to a second target or may not specifically or preferentially bind to a second target. As such, "immunospecific binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means "immunospecific" binding. Natural antibodies are only capable of binding to one epitope species (i.e., they are "monospecific"), although they may immunospecifically bind to multiple copies of that species (i.e., exhibit "bivalent" or "multivalent"). Two molecules are considered to be capable of binding to each other in a "physiologically specific" manner if such binding exhibits the specificity of the receptors binding to their respective ligands.

A resurgence Of interest In The therapeutic potential Of Antibodies has been seen over The past decades and Antibodies have become one Of The leading classes Of biotechnologically derived drugs (Chan, c.e. et al (2009) "The Use Of Antibodies In The Treatment Of infection Diseases," Singapore med.j.50(7): 663-666). Over 200 antibody-based drugs have been approved for use or in development.

1. General structural Properties of antibodies

The basic building block of naturally occurring immunoglobulins (e.g., IgG) is a tetramer consisting of two shorter "light chains" complexed with two longer "heavy chains" and is typically expressed as a glycoprotein of about 150,000 Da. Each chain is composed of an amino-terminal ("N-terminal") portion comprising a "variable domain" and a carboxy-terminal ("C-terminal") portion comprising at least one "constant domain". An IgG light chain consists of a single "light chain variable domain" ("VL") and a single "light chain constant domain" ("CL"). Thus, the structure of the light chain of an IgG molecule is N-VL-CL-C (where N and C represent the N-terminus and C-terminus of the polypeptide, respectively). An IgG heavy chain consists of a single "heavy chain variable domain" ("VH"), three "heavy chain constant domains" ("CH 1", "CH 2", and "CH 3"), and a "hinge" region ("H") located between the CH 1and CH2 domains. Thus, the IgG heavy chain has the structure N-VH-CH1-H-CH2-CH3-C (where N and C represent the N-and C-termini of the polypeptide, respectively). The ability of an intact, unmodified antibody (e.g., an IgG antibody) to bind an epitope of an antigen depends on the presence and sequence of the variable domains. Unless specifically stated otherwise, the sequence of domains of the protein molecules described herein is in the "N-terminal to C-terminal" orientation.

(a) Constant domains

(i) Light chain constant domains

Preferred CL domains are human IgG clk domains. An exemplary amino acid sequence of the human CL κ domain is (SEQ ID NO: 1):

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

alternatively, an exemplary CL domain is a human IgG CL λ domain. An exemplary amino acid sequence of the human CL λ domain is (SEQ ID NO: 2):

QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS

(ii) heavy chain CH1 Domain

The CH1 domains of the two heavy chains of an antibody are complexed with the "CL" constant region of the light chain of the antibody and are attached to the CH2 domain of the heavy chain via an intervening hinge domain.

An exemplary CH1 domain is the human IgG1 CH1 domain. The amino acid sequence of the exemplary human IgG1 CH1 domain is (SEQ ID NO: 3):

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV

an exemplary CH1 domain is the human IgG2 CH1 domain. The amino acid sequence of the exemplary human IgG2 CH1 domain is (SEQ ID NO: 4):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV

an exemplary CH1 domain is the human IgG3 CH1 domain. The amino acid sequence of the exemplary human IgG3 CH1 domain is (SEQ ID NO: 5):

ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV

an exemplary CH1 domain is the human IgG4 CH1 domain. The amino acid sequence of the exemplary human IgG4 CH1 domain is (SEQ ID NO: 6):

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

(b) heavy chain hinge region

One exemplary hinge domain is the human IgG1 hinge domain. The amino acid sequence of the hinge domain of exemplary human IgG1 is (SEQ ID NO: 7): EPKSCDKTHTCPPCP.

Another exemplary hinge domain is the human IgG2 hinge domain. The amino acid sequence of the hinge domain of exemplary human IgG2 is (SEQ ID NO: 8): ERKCCVECPPCP are provided.

Another exemplary hinge domain is the human IgG3 hinge domain. The amino acid sequence of the hinge domain of exemplary human IgG3 is (SEQ ID NO: 9):

ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP。

another exemplary hinge domain is the human IgG4 hinge domain. The amino acid sequence of the hinge domain of exemplary human IgG4 is (SEQ ID NO: 10): ESKYGPPCPSCP are provided. As described herein, the IgG4 hinge domain may include a stabilizing mutation such as an S228P substitution. Exemplary S228P-amino acid sequence of the hinge domain of stabilized human IgG4 is (SEQ ID NO: 11): ESKYGPPCPPCP are provided.

(c) Heavy chain CH2 and CH3 domains

The CH2 and CH3 domains of the two heavy chains interact to form the "Fc domain" of an IgG antibody that is recognized by cellular Fc receptors, including but not limited to Fc γ receptors (Fc γ R). As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. A portion of an Fc region (including portions encompassing the entire Fc region) is referred to herein as an "Fc domain". An Fc region is considered to have a particular IgG isotype, class, or subclass if its amino acid sequence is most homologous to that isotype relative to other IgG isotypes. In addition to their known uses in diagnostics, antibodies have also been shown to be useful as therapeutic agents.

The amino acid sequence of the CH2-CH3 domain of exemplary human IgG1 is (SEQ ID NO: 12):

as numbered by the EU index shown in Kabat, whereinXIs lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of exemplary human IgG2 is (SEQ ID NO: 13):

as numbered by the EU index shown in Kabat, whereinXIs lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of exemplary human IgG3 is (SEQ ID NO: 14):

as numbered by the EU index shown in Kabat, whereinXIs lysine (K) or absent.

The amino acid sequence of the CH2-CH3 domain of exemplary human IgG4 is (SEQ ID NO: 15):

as numbered by the EU index shown in Kabat, whereinXIs lysine (K) or absent.

Throughout the present specification, the numbering of residues in the constant region of an IgG heavy chain is that according to the EU index in Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service (Public Health Service), NH1, MD (1991) ("Kabat"), which is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of the constant domains of the human IgG1 EU antibody. Polymorphisms have been observed at a number of different positions within the antibody constant region (e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 numbered by the EU index as shown in Kabat), and thus there may be slight differences between the sequences displayed and those in the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18Gm allotypes are known: g1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, G1, c5, u, v, G5) (Lefranc et al, "The Human IgG Subclases: Molecular Analysis Of Structure, Function And Regulation," Pergamon, Oxford, pp.43-78 (1990); Lefranc, G. et al 9, hum. Gene et al 50, 197199). In particular, it is contemplated that the antibodies of the invention may incorporate any allotype (allotype), allotype (isoallotype), or haplotype (haplotype) of any immunoglobulin gene, and are not limited to the allotypes, or haplotypes of the sequences provided herein. Furthermore, in some expression systems, the C-terminal amino acid residue of the CH3 domain (bold above) may be removed post-translationally. Accordingly, in the gp 41-binding molecules of the invention, the C-terminal residue of the CH3 domain is an optional amino acid residue. Specifically, encompassed by the present invention are gp 41-binding molecules that lack the C-terminal residue of the CH3 domain. Also specifically encompassed by the present invention are such constructs comprising the C-terminal lysine residue of the CH3 domain.

(d) Variable domains

The variable domain of an IgG molecule consists of three "complementarity determining regions" ("CDRs") comprising amino acid residues of an antibody that will contact an epitope, and intervening non-CDR segments called "framework regions" ("FRs") that substantially maintain the structure of the CDR loops (loops) and position the CDR loops to allow such contact (although some framework residues may also contact the epitope). Thus, the VL domain and the VH domain have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4-c. The amino acid sequence of the CDRs determines whether the antibody will be able to bind a particular epitope. The interaction of antibody light chains with antibody heavy chains, and in particular, the interaction of their VL and VH domains, forms the epitope-binding domain of the antibody.

Amino acids from the variable domains of mature heavy and light chains of immunoglobulins are also named by the position of the amino acid in the chain. Kabat describes a number of amino acid sequences for an antibody, identifies the amino acid consensus sequence for each subgroup and assigns a residue number to each amino acid, and identifies CDRs, as defined by Kabat (it is understood that by Chothia, C).&Lesk, A.M. ((1987) "immunologic Structures For The Hypervariable Regions Of immunologlobulins," J.mol.biol.196:901-_H1 starts five residues earlier). The numbering scheme of Kabat may be byThe antibodies considered were aligned with one of the consensus sequences in Kabat with reference to conserved amino acids and extended to antibodies not included in their schema. This method for assigning residue numbers has become standard in the art and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of the human antibody light chain occupies a position equivalent to the amino acid at position 50 of the mouse antibody light chain.

Polypeptides that are (or can be used as) the first CDR, the second CDR, and the third CDR of an antibody light chain are designated herein as: CDR_L1 Domain, CDR_L2 Domain and CDR_L3 domain. Similarly, polypeptides that are (or can be used as) the first CDR, the second CDR, and the third CDR of an antibody heavy chain are designated herein as: CDR_H1 Domain, CDR_H2 Domain and CDR_H3 domain. Thus, the term CDR_L1 Domain, CDR_L2 Domain, CDR_L3 Domain, CDR_H1 Domain, CDR_H2 Domain and CDR_H3 domain relates to such a polypeptide: when incorporated into a protein, causes the protein to be capable of binding a particular epitope, whether such protein is an antibody having a light chain and a heavy chain or a diabody or a single chain binding molecule (e.g., scFv, IgG, and IgG, respectively,Etc.), or another type of protein.

The term "epitope binding domain" (e.g., a domain that binds gp 41) means a portion of a binding molecule (or a polypeptide having the amino acid sequence of such a portion) that contributes to the ability of the binding molecule to immunospecifically bind to an epitope (e.g., an epitope of gp 41). The epitope binding domain may comprise the VL domain or the VH domain of an antibody, or any 1, 2, 3,4 or 5 CDR domains of an antibody, or may comprise all 6 CDR domains of an antibody, and, while capable of immunospecific binding to such an epitope, may exhibit immunospecificity, affinity or selectivity for such an epitope other than that of such an antibody. The epitope binding domain may contain only a portion of the CDRs,namely, for the subset Of CDR Residues required for binding, the term SDR (Kim, J.H. et al (2012) "mutation By CDR-Grafting And Specificity-Determining Residue-Grafting," Methods mol. biol.907: 237-245; Kim, K.S. et al (2010) "Construction Of A mutation By To therapy B Surface antibiotic By Specificity-Determining Residues B mutation By mutation-Determining Residues (SDR) -Grafting Residue-Grafting," biochemistry. Biophys. Res. Commun.396(2): 231-237; Kashmiri, S.V. et al (2005) "mutation-A New application restriction Residue-modification," method 19-36. N. 25. 34. mutation. N.8651. Grafting "mutation-A mutation-N.8641. PCR amplification By mutation-A mutation-modification". Preferably, however, the epitope binding domain will contain all 6 CDR domains of such an antibody. The epitope binding domain can be a single polypeptide chain (e.g., scFv), or can include two or more polypeptide chains, each of which can have an amino terminus and a hydroxyl terminus (e.g., diabody, Fab portion₂Moieties, etc.) and they may be covalently bound to each other via disulfide bonds.

2. Humanization of antibodies

The invention also specifically encompasses binding molecules comprising either the VL domain or the VH domain of an antibody, and preferably both the VL domain and the VH domain of an antibody. Preferably, such an antibody is a humanized antibody. Monoclonal antibodies are typically prepared in non-human species such as mice or rabbits. The variable and/or constant domains of such antibodies can be recognized as immunogens, thereby eliciting an immune response against them. However, such molecules may be "humanized" by introducing one or more amino acid substitutions to make such antibodies more like those produced by humans, thereby reducing or eliminating their immunogenicity. The term "humanized" antibody refers to a chimeric molecule, typically prepared using recombinant techniques, that has an epitope binding site of an immunoglobulin from a non-human species and the immunoglobulin structure of the remaining molecule based on the structure and/or sequence of a human immunoglobulin. The polynucleotide sequences of the variable domains of such antibodies can be used for genetic manipulation to produce such derivatives and to improve the affinity or other characteristics of such antibodies. Has been prepared by LoBuglio, A.F. et al (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: kinetics And Immune Response, "Proc. Natl. Acad. Sci. (U.S.A.) -86: 4220-4224; sato, K. et al (1993) Cancer Res 53:851-856.Riechmann, L. et al (1988) "rehaping Human Antibodies for Therapy," Nature332: 323-; verhoeyen, M. et al (1988) "rehaping Human Antibodies: grafting An anticoagulant Activity, "Science 239: 1534-; kettleborough, C.A. et al (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: the impedance Of Framework resources On Loop conversion, "Protein Engineering4: 773-; maeda, H.et al (1991) "Construction Of repaired Human Antibodies With HIV-neutralling Activity," Human Antibodies hybrids 2: 124-; gorman, S.D. et al (1991) "Reshaping A Therapeutic CD4 Antibody," Proc.Natl.Acad.Sci. (U.S.A.)88: 4181-4185; tempest, P.R. et al (1991) "rehaping A Human Monoclonal Antibody To Inhibit Human Respiratory synthesis Infection in vivo," Bio/Technology 9: 266-; co, M.S. et al (1991) "manipulated Antibodies For antibody Therapy," Proc.Natl.Acad.Sci. (U.S.A.)88: 2869-2873; carter, P.et al (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.)89: 4285-4289; and Co, M.S. et al (1992) "Chinese And human Antibodies With Specificity For The CD33 antibody," J.Immunol.148: 1149-. In some embodiments, the humanized antibody retains all of the CDR sequences (e.g., a humanized mouse antibody comprising all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more (one, two, three, four, five or six) CDRs that differ in sequence relative to the original antibody.

The general principle of humanizing an antibody involves retaining the basic sequence of the epitope-binding domain of the antibody while exchanging the non-human remainder of the antibody with human antibody sequences. There are four general procedures for humanizing monoclonal antibodies. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light chain variable domain and heavy chain variable domain; (2) designing a humanized or caninized antibody, i.e., determining which antibody framework regions to use during the humanization or caninization process; (3) actual humanization or caninization methods/techniques; and (4) transfection and expression of humanized antibodies. See, for example, U.S. Pat. nos. 4,816,567, 5,807,715, 5,866,692, and 6,331,415.

A number Of humanized Antibody molecules comprising epitope binding domains derived from non-Human immunoglobulins have been described, including Chimeric Antibodies having rodent variable domains or modified rodent variable domains And their Associated Complementarity Determining Regions (CDRs) fused To Human constant domains (see, e.g., Winter et al (1991) "Man-male Antibodies," Nature 349: 293-299; Lobuglio et al (1989) "Mouse/Human Chimeric Antibody In Man: kinetic Antibody Response," Proc. Natl.Acad.Sci. (U.S.A.; 86:4220-4224 (1989); Shaw et al (1987) "Characterification Of A molecular Antibody (17-1A) ToTuTuTumouth Antibody-scientific;" molecular Antibody 45138: Monoclonal Antibody 45138 And Cancer 3547: biological Antibody et al (1989). Other references describe rodent CDRs which are grafted into The Human Framework Region (FR) prior to fusion With The appropriate Human Antibody constant domains (see, e.g., Riechmann, L. et al (1988) "rehaping Human Antibodies for Therapy," Nature332: 323-. Another reference describes rodent CDRs supported by recombinantly modified rodent framework regions. See, for example, european patent publication No. 519,596. These "humanized" molecules are designed to minimize the undesirable immune response to rodent anti-human antibody molecules that limits the duration and efficacy of therapeutic applications of those moieties in human recipients. Other methods of humanizing antibodies that may also be used are disclosed in the following documents: daugherty et al (1991) "Polymerase Chain Reaction facilities The Cloning, CDR-Grafting, And d Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18Component Of Leukocyte antigens," Nucl. acids Res.19: 2471-. The invention specifically encompasses binding molecules (including antibodies and diabodies) comprising the VL domain and/or VH domain of a "humanized" antibody.

Despite this success, the preparation of stable, functional heterodimeric, non-monospecific diabodies optimized for therapeutic use can be further improved by careful consideration and placement of the domains employed in the polypeptide chains. Thus, the present invention relates to providing specific polypeptides specifically designed to be covalently bound to form stable and therapeutically useful heterodimeric diabodies and heterodimeric Fc diabodies capable of simultaneously binding gp41 and a molecule present on the surface of an immune effector cell.

B. Bispecific antibodies

As indicated above, natural antibodies are capable of binding to only one epitope species (i.e., they are monospecific), although they may bind to multiple copies of that species (i.e., display bivalency or multivalency). The ability of an antibody to bind an epitope of an antigen depends on the presence and amino acid sequence of the VL and VH domains of the antibody. The interaction of the light and heavy chains of an antibody and, in particular, the interaction of its VL and VH domains forms one of the two epitope-binding domains of a natural antibody, such as IgG.

The functionality of an antibody can be enhanced by generating a multispecific antibody-based molecule that can simultaneously bind two separate and distinct antigens (or different epitopes of the same antigen) and/or by generating an antibody-based molecule that has a higher valency (i.e., greater than two binding domains) for the same epitope and/or antigen.

In order to provide molecules with higher capacity than natural antibodies, various recombinant bispecific antibody formats have been developed (see, e.g., PCT publications WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/018386, WO 2012/009544, WO 2013/070565). Most such methods use linker peptides to fuse or fuse additional binding domains (e.g., scFv, VL, VH, etc.) to an antibody core (IgA, IgD, IgE, IgG, or IgM) or to fuse multiple antibody binding moieties (e.g., two Fab portions or scFv) to one another. Alternative forms use linker peptides to fuse binding proteins (e.g., scFv, VL, VH, etc.) to dimeric domains such as CH2-CH3 domains or alternative polypeptides (WO 2005/070966, WO 2006/107786A, WO 2006/107617A, WO 2007/046893). Typically, this approach involves a compromise (compromises) and a trade-off (trade-off of fs). For example, PCT publications WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose that the use of linkers may cause problems in therapeutic settings and teach such trispecific antibodies: wherein the CL domain and CH1 domain have been switched from their natural positions and the VL domain and VH domain have been diversified (WO 2008/027236, WO 2010/108127) to allow them to bind more than one antigen. Thus, the molecules disclosed in these documents trade binding specificity for the ability to bind to additional antigen species. PCT publications WO 2013/163427 and WO 2013/119903 disclose that the CH2 domain is modified to include a fusion protein adduct (adduct) that includes a binding domain. The document indicates that the CH2 domain may play only a minimal role in mediating effector function. PCT publications WO 2010/028797, WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc regions have been replaced with additional VL and VH domains to form trivalent binding molecules. PCT publication nos. WO 2003/025018 and WO2003012069 disclose recombinant diabodies whose single chains comprise scFv domains. PCT publication No. WO 2013/006544 discloses multivalent Fab molecules that are synthesized as a single polypeptide chain and then undergo proteolysis to produce a heterodimeric structure. Thus, the molecules disclosed in these documents trade all or some of the ability to mediate effector functions for the ability to bind additional antigenic species. PCT publication nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose the addition of additional binding domains or functional groups to an antibody or antibody portion (e.g., the addition of a diabody to the light chain of an antibody, or the addition of additional VL and VH domains to the light and heavy chains of an antibody, or the addition of a heterologous fusion protein or linking multiple Fab domains to each other). Thus, the molecules disclosed in these documents trade natural antibody structure for the ability to bind additional antigen species.

Bispecific diabodies

The ability To generate Diabodies other than natural antibodies in terms of being able To bind two or more different epitope species (i.e.exhibiting Bispecific or multispecific properties in addition To Bivalent or multivalent) has also been indicated in The art (see, e.g., Holliger et al (1993) 'Diabodies': Small Bivalent And Bispecific Antibody Fragments, "Proc. Natl. Acad. Sci. (U.S. A.)90: 6444. 6448; US 2004/0058400(Hollinger et al); US 2004/0220388(Mertens et al); Alt et al (1999) FEBS letter.454 (1-2): 90-94; Lu, D. et al (2005)" A Fully Recombinant IgG-Like Bispecific Antibody Toth thermal IgG-phase Growth of molecular Growth, cellulose, molecular Growth Applications, "Protein Eng Des Sel.17(1): 21-27; wu, A. et al (2001) "multimedia Of A chiral Anti-CD20 Single Chain Fv-Fv Fusion Protein a media Through Variable Domain Exchange," Protein Engineering 14(2): 1025) 1033; asano et al (2004) "A diabetes For Cancer Immunotherapy And Its Of Human Fc Domain," Abstract 3P-683, J.biochem.76(8): 992; takemura, S. et al (2000) "Construction Of A diabetes Using A refining System," Protein Eng.13(8): 583-; baeuerrle, P.A. et al (2009) "Bispecific T cell Engaging Antibodies For Cancer Therapy," Cancer Res.69(12): 4941-.

The design of diabodies is based on the structure of single chain variable domain parts (scfvs), where light and heavy chain variable domains are linked to each other using short linking peptides. Bird et al (1988) ("Single-Chain antibody-Binding Proteins," Science 242:423-426) describe examples of linker peptides that bridge about 3.5nm between the carboxy terminus of one variable domain and the amino terminus of another variable domain. Linkers of other sequences have been designed and used (Bird et al (1988) "Single-Chain antibody-Binding Proteins," Science 242: 423-. The linker may in turn be modified for additional functions, such as attachment of a drug or attachment to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, an appropriate polynucleotide-containing plasmid encoding scFv can be introduced into an appropriate host cell, a eukaryotic cell, such as a yeast, plant, insect or mammalian cell; or prokaryotic cells such as E.coli. Polynucleotides encoding the scFv of interest can be made by conventional procedures such as ligation (ligation) of the polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

Providing non-monospecific "diabodies" brings significant advantages for antibodies: the ability to co-ligate and co-localize cells expressing different epitopes. Thus, bispecific diabodies have a wide range of applications including therapy and immunodiagnostics. Bispecific allows great flexibility in the design and engineering of diabodies in various applications, providing enhanced affinity for multimeric antigens, cross-linking of different antigens, and targeted targeting of specific cell types depending on the presence of both target antigens. Diabody molecules known In the art have also shown particular utility In the field of tumor imaging due to their bivalent, low off-rate and rapid clearance from circulation (equal to or below-50 kDa for small-sized Diabodies) (Fitzgerald et al (1997) "Improved tumor Targeting By lipid Stabilized Diabodies Expressed In Pichia pastoris" Protein Eng.10: 1221).

The ability To generate Bispecific diabodies has led To their use (in "trans)") For co-linking two Cells together, For example, By co-linking receptors present on the surface Of different Cells (e.g., cross-linking Cytotoxic T-Cells To Target Cells such as cancer Cells expressing disease antigens or Cells Infected with pathogens) (Staerz et al (1985) "Hybrid Antibodies Can Target Sites T Cells For attached Cells," Nature 314: 628. 631. and Holliger et al (1996) "Specific kits Of lymphoid Cells B Cytotoxic T-Cells Mediated B A biological reagents," Protein Eng.9: 299. Cells 305; vitamin et al (2005) "binding applications To IgG-lipid Antibodies," HIV drug proteins cell 5. Mar. 9: 299. sub. 12. cell proteins) cell binding proteins B cell III. sub. 5. 12. K. sub. K. sub. K. sub. K. sub. K. No. 5. K. sub. K. sub. cell No. 5. K. sub. K. cell No. 5. K. sub. 3. K. Cells, HIV strain, "PLoS Patholog 11(11): e1005233.doi:10.1371/journal. ppat.1005233)). Alternatively (or additionally), bispecific (or multispecific) diabodies can be used to co-link (in "cis") molecules, such as receptors, etc., that are present on the surface of the same cell. Co-linking of different cells and/or receptors is used to modulate effector function and/or immune cell signaling. Multispecific Molecules (e.g., bispecific diabodies) comprising epitope binding domains may be directed against surface determinants Of any immune Cell expressed on T lymphocytes, Natural Killer (NK) cells, antigen presenting cells or other monocytes, such as CD2, CD3, CD8, CD16, TCR, NKG2D etc., or against surface determinants Of B cells, such as CD19, CD20, CD22, CD30, CD37, CD40 and CD74(Moore, P.A. et al (2011) "Application Of Dual Affinity targeting Molecules To Achie Optimal modified T Cell decorating Of B-Lymphoma," Blood117(17):4542 4551; Cheson, B.D. et al (2008) Monoclonal Antibody Therapy B-Cell forge J-41, Cell J.31., "Cell 65. 31, 23.31.," Cell J.11. "(2008): Cell) 13. 23. 31, Cell J.11, et al (2008). In particular, epitope binding domains directed against cell surface receptors present on immune effector cells are used to generate multispecific binding molecules capable of mediating redirected cell killing.

In many studies, diabodies that bind to determinants Of effector Cells, e.g., Fc gamma receptor (Fc gamma R), have also been found to activate effector Cells (Holliger et al (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells media By A Bispecific Diabody," Protein Eng.9: 299-305; Holliger et al (1999) "Carcinometric antibody (CEA) -Specific T-cell Activation In Colon Carcinoma Induced By By Anti-CD 3x Anti-CEA Bispecific Diabody B7 x Anti-CEA Specific Fusion Proteins," Cancer Res.59:2909-2916 And PCT publication No. WO 36 2006/113665; WO 2010/080538; WO 46 2012/162068). Typically, effector cell activation is triggered by the binding of an antibody that binds an antigen to an effector cell via Fc-fcyr interaction; thus, in this regard, diabody molecules can exhibit Ig-like functionality independent of whether they include an Fc domain (e.g., as analyzed in any effector function test (e.g., ADCC test) known in the art or exemplified herein). By cross-linking tumor cells and effector cells, diabodies not only bring effector cells into the vicinity of tumor cells, but also lead to effective tumor killing (see, e.g., Cao et al (2003), "Bispecific Antibody Conjugates In Therapeutics," adv. drug. Deliv. Rev.55: 171-.

However, the cost of the advantages of the bispecific diabodies described above is significant. The formation of such non-monospecific diabodies requires the successful assembly of two or more distinct and distinct polypeptides (i.e., such formation requires the formation of diabodies through heterodimerization of different polypeptide chain species). This fact is in contrast to monospecific diabodies formed by homodimerization of the same polypeptide chains. Since at least two different polypeptides (i.e.two polypeptide species) must be provided in order to form a non-monospecific Diabody, and since the homodimerization Of such a polypeptide results in an inactive molecule (Takemura, S.et al (2000) "Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A reflex System," Protein Eng.13(8): 583. sup. 588), the production Of such a polypeptide must be done in a manner that prevents covalent binding between polypeptides Of the same species (i.e.in order to prevent homodimerization) (Takemura, S.et al (2000 Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A reflex, "Protein Eng.13(8): 583. sup. 588). Thus, The art has taught non-Covalent association Of such polypeptides (see, For example, Olafsen et al (2004) "proportional Disf-Linked Anti-CEA diagnostic Allowss Site-Specific Conjugation And radial Targeting Applications," put. Engr. Des. Sel.17: 21-27; Asano et al (2004) "A diabetes For Cancer Immunotherapy And Its Functional Enhancement B.F.main," Abstract 3P-683, J.Biond.76 (8): 992; Takemura, S. dy et al (2000) "Conjugation A diabetes (Small scientific Anti biological Antibody) binding A.1962; chemical modification A. filtration, S. 10. reaction, J.11) And" reaction family reaction J.9. reaction, Protein, J.65. (72) biological binding, reaction.

However, it is recognized in The art that Bispecific diabodies composed of non-covalently associated polypeptides are unstable And readily dissociate into non-functional single polypeptide chain monomers (see, e.g., Lu, D. et al (2005) "A Fully Human Recombinant IgG-Like Bispecific Antibody To The organism The epidemic Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antibody Activity," J.biol.Chem.280(20): 19665-19672).

Facing this challenge, the art has successfully developed stable covalently bound heterodimeric non-monospecific diabodies, termed heterodimericDiabodies, see, for example, Liu.L et al (2017) "MGD 011, A CD19x CD3 Dual-Affinity targeting Bi-specific molecular incorporation Extended circulation Half-life for the Treatment of B-Cell Malignances," Clin Cancer Res.23(6): 1506-; tsai, P.et al (2016) "CD 19xCD3 DART proteins Human B-Cell deletion In Vivo In Humanized BLT Rice," mol.ther. Oncolyytics 3:15024. doi: 10.1038/mt.2015.24; chen, X, et al (2016) "mechanical project of First-in-Human Dose for Bispecific immunological modellingtory P-Cadherin LP-DART: an Integrated PK/PD Modeling Approach, "Clin. Pharmacol. Ther.100(3): 232-241; sloan, D.D. et al (2015) "Targeting HIV Reservoir in Infected CD 4T Cells by Dual-Affinity Re-Targeting Molecules (DARTs) that bit HIV Envelope and Recircuit cytoxic T Cells," PLoS Patholog.11 (11): e1005233.doi: 10.1371/journal.ppat.1005233; al Hussaini, M. et Al (2015) "Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-TargetingPlatform, "Blood pii: blood-2014-05-575704; chichili, G.R. et al (2015) "A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenuses Leukemia: (iii) preferably Activity And Safety In non human formats, "Sci. Transl. Med.7(289):289ra 82; johnson, S.et al (2010) "Effect Cell Recirculation With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Point Tumor Cytolysis And In Vivo B-Cell deletion," J.Molec.biol.399(3): 436-) -449; veri, M.C. et al (2010) "Therapeutic Control Of B Cell Activation Via Recirculation Of Fcgamma Receptor IIB (CD32B) inhibition Function With A Novel binary Antibody Scaffold," Arthritis Rheum.62(7): 1933-1943; moore, P.A. et al (2011) "Application Of Dual Affinity targeting Molecules To Achieve Optimal Redirected T Cell Kiling Of B-Cell Lymphoma," Blood117(17): 4542-4551; U.S. patent nos. 9,932,400; 9,908,938, respectively; 9,889,197, respectively; 9,884,921, respectively; 9,822,181, respectively; 9,296,816, respectively; 9,284,375, respectively; 8,795,667, respectively; 8,187,593, respectively; 8,669,349, respectively; 8,784,808, respectively; 8,795,667, respectively; 8,802,091, respectively; 8,802,093, respectively; 8,946,387 and 8,968,730, and U.S. patent publication No. 2009/0060910; 2010/0174053, respectively; 2011/0081347, respectively; 2011/0097323, respectively; 2011/0117089, respectively; 2012/0034221, respectively; 2012/0294796, respectively; 2013/0149236, respectively; 2013/0295121, respectively; 2014/0017237, respectively; 2014/0099318, respectively; 2014/0255407, respectively; 2015/0175697, respectively; 2016/0017038, respectively; 2016/0159908, respectively; 2016/0159908, respectively; 2016/0194396, respectively; 2016/0194396, respectively; 2016/0200827, respectively; 2016/0200827, respectively; 2016/0222105, respectively; 2016/0222105, respectively; 2016/0333111, respectively; 2016/0355586, respectively; 2016/0355586, respectively; 2017/0157251, respectively; 2017/0157251, respectively; 2017/0198037, respectively;2017/0198037, respectively; 2017/0198045, respectively; 2017/0204176, respectively; 2017/0204176, respectively; 2017/0247452 and 2018/0094072; european patent document No. EP 1868650; EP 2158221; EP 2247304; EP 2252631; EP 2282770; EP 2328934; EP 2376109; EP 2542256; EP 2601216; EP 2714079; EP 2714733; EP 2786762; EP 2839842; EP 2840091; and PCT publication No. WO 2006/113665; WO 2008/157379; WO 2010/027797; WO 2010/033279; WO 2010/080538; WO 2011/109400; WO 2012/018687; WO 2012/162067; WO 2012/162068; WO 2014/159940; WO 2015/021089; WO 2015/026892 and WO 2015/026894). Such diabodies comprise two or more covalently complexed polypeptides and involve engineering one or more cysteine residues into each employed polypeptide species that allow for the formation of disulfide bonds and thereby covalently bind one or more pairs of such polypeptide chains to each other. For example, it has been shown that cysteine residues are added to the C-terminus of such constructs to allow disulfide bonding between the involved polypeptide chains, thereby stabilizing the resulting diabodies and not interfering with the binding characteristics of the diabodies.

The simplestDiabodies comprise two polypeptide chains, each comprising three domains (fig. 1A-fig. 1B). The first polypeptide chain comprises: (i) a domain comprising a binding region for a light chain variable domain of a first immunoglobulin (VL1), (ii) a second domain comprising a binding region for a heavy chain variable domain of a second immunoglobulin (VH2), and (iii) a third domain to promote heterodimerization with a second polypeptide chain and to covalently bind a first polypeptide chain of a diabody to a second polypeptide chain ("heterodimer promotion domain"). The second polypeptide chain comprises a complementary first domain (VL2 domain), a complementary second domain (VH1 domain), and a third domain that complexes with the third domain of the first polypeptide chain to promote heterodimerization and covalent binding to the first polypeptide chain ("heterodimer promoting domain"). Such molecules are stable, potent and have the ability to bind two or more antigens simultaneously. In one embodiment, the first polypeptide chain and the second polypeptide chainThe third domains each comprise a cysteineResidues for binding polypeptides together via disulfide bonds.

The third domain of one or both of the polypeptide chains can additionally have the sequence of CH2-CH3 domain such that complexation of the diabody polypeptide forms an Fc domain capable of binding to an Fc receptor of a cell (e.g., B lymphocyte, dendritic cell, natural killer cell, macrophage, neutrophil, eosinophil, basophil, and mast cell). Many variations of this molecule have been described (see, e.g., U.S. patent publication nos. 2013-0295121; 2010-0174053; 2007-0004909; 2009-0060910; european patent publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT publication No. WO 2012/162068; WO 2012/018687; WO 2010/080538; WO 2006/113665) and are provided herein. Many variations of this molecule have been described (see, e.g., U.S. patent publication Nos. 2015/0175697; 2014/0255407; 2014/0099318; 2013/0295121; 2010/0174053; 2009/0060910; 2007) 0004909; European patent publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221; EP 1868650 and PCT publication No. WO 2012/162068; WO 2012/018687; WO 2010/080538; WO 2006/113665) and are provided herein.

For applications where bispecific or tetravalent molecules are desired but no Fc is required, alternative constructs are known in the art, including but not limited to bispecific T cell engaging molecules, also known as Fc(see, e.g., PCT publication Nos.: WO 1993/11161 and WO 2004/106381) and tetravalent tandem antibodies, also known as(see, for example, U.S. patent publication No.: 2011-one 0206672; European patent publication No. EP 2371866 and PCT publications No. WO 1999/057150, WO 2003/025018 and WO 2013/013700).Formed from a single polypeptide chain comprising serially linked scFvsFormed by homodimerization of two identical polypeptide chains, each provided with a VH1 domain, a VL2 domain, a VH2 domain and a VL2 domain.

Embodiments of the invention

The present invention relates to "epitope-binding molecules" comprising "epitope-binding domains", such as CDRs_L1 Domain, CDR_L2 Domain, CDR_L3 Domain, CDR_H1 Domain, CDR_H2 Domain or CDR_H3 domain, or any combination or subcombination thereof sufficient to form an "epitope-binding site" that allows the molecule to bind to an epitope. For example, a "molecule that binds an epitope of gp 41" includes a "domain that binds an epitope," such as a CDR_L1 Domain, CDR_L2 Domain, CDR_L3 Domain, CDR_H1 Domain, CDR_H2 Domain or CDR_H3 domain, or any combination or subcombination thereof sufficient to form a "site binding an epitope of gp 41" that allows the molecule to bind an epitope of gp 41. Preferably, however, such a gp 41-binding molecule will possess the CDRs of an "anti-gp 41 antibody" that immunospecifically binds to an epitope of gp41_L1 Domain, CDR_L2 Domain, CDR_L3 Domain, CDR_H1 Domain, CDR_H2 Domain and CDR_H3 domain. In some embodiments, such molecules may include only the gp41-VL binding domain of such an anti-gp 41 antibody or only the gp41-VH binding domain of such an anti-gp 41 antibody, which domains may interact with other binding domains to mediate immunospecific binding to an epitope of gp41, or may be sufficient to mediate such binding by themselves. In some embodiments, such molecules can include both the gp41-VL binding domain of an anti-gp 41 antibody and the gp41-VH binding domain of an anti-gp 41 antibody, which can be derived from the same antibody or different antibodies.

According to the inventionOne embodiment relates to a molecule that "multispecific" binds gp41, is bispecific and is capable of binding a "first epitope" and a "second epitope", which epitopes are not identical to each other. The representation of an epitope as "first epitope", "second epitope" etc. is only intended to provide a prerequisite basis for their description and does not imply a substantial difference. Such multispecific molecules include a "VL 1" domain/"VH 1" domain capable of binding a first epitope, and a "VL 2" domain/"VH 2" domain capable of binding a second epitope. The symbols "VL 1" and "VH 1" represent the variable light chain domain and variable heavy chain domain, respectively, that bind the first epitope of such bispecific molecules. Similarly, the symbols "VL 2" and "VH 2" denote the light chain variable domain and heavy chain variable domain, respectively, that bind the second epitope of such bispecific molecules. It does not matter whether a particular epitope is designated as a first epitope, a second epitope, etc.; such symbols have only a relationship with respect to the presence and orientation of the domains of the polypeptide chain of the binding molecule of the invention. In one embodiment, one such epitope is an epitope of human gp41, and another such epitope is a different epitope of gp41 or an epitope of a molecule that is not gp 41. In particular embodiments, one such epitope is an epitope of human gp41, and another such epitope is an epitope of a molecule (e.g., CD2, CD3, CD8, CD16, T-cell receptor (TCR), NKG2D, etc.) that is present on the surface of an effector cell, such as a T lymphocyte, Natural Killer (NK) cell, or other monocyte. In certain embodiments, the multispecific molecule comprises more than two epitope binding domains. In certain embodiments, the multispecific molecules of the present invention are trispecific and capable of binding a first epitope, a second epitope and a "third epitope". Such a trispecific molecule comprises a VL1 domain/VH 1 domain capable of binding a first epitope, a VL2 domain/VH 2 domain capable of binding a second epitope and "VL 3" and "VH 3" domains capable of binding a third epitope. In other embodiments, the multispecific molecules of the present invention are tetraspecific and capable of binding to a first epitope, a second epitope, a third epitope and a "fourth epitope". Such tetraspecific molecules comprise a VL1 domain capable of binding a first epitopea/VH 1 domain, a VL2 domain/VH 2 domain capable of binding a second epitope, a VL3 domain/VH 3 domain capable of binding a third epitope, and a "VL 4" domain/"VH 4" domain capable of binding a fourth epitope. Such multispecific molecules will bind at least one epitope of gp41 and at least one epitope of a molecule other than gp41, and may further bind another epitope of gp41 and/or another epitope of a molecule other than gp 41. The invention specifically encompasses bispecific diabodies, produced using any of the methods provided herein,Antibody, antibody,And trivalent molecules.

Molecules that multispecific bind gp41

In one embodiment, a gp 41-binding molecule of the invention will be a multispecific gp 41-binding molecule capable of binding two or more different epitopes and will include:

(1) a first epitope binding domain that immunospecifically binds to an epitope of gp41 (i.e., binds to a domain of gp41, preferably comprising an optimized gp 41-binding VL domain of the invention and/or an optimized gp 41-binding VH domain of the invention); and

(2) a second epitope binding domain that immunospecifically binds a second epitope, wherein such second epitope is (i) a different epitope of gp41 or (ii) an epitope of a molecule other than gp 41; and

(3) optionally, one or more additional epitope binding domains that immunospecifically binds a third additional epitope, and a fourth additional epitope, etc., wherein such additional epitope binding domains can be the same or different from the first epitope or the second epitope.

Such molecules that bind gp41 preferably include a combination of epitope binding domains that recognize a set of antigens unique to a target cell or immune effector cell. In particular, the present invention relates to molecules that multi-specifically bind gp41, which are capable of binding to an epitope of gp41 and to an epitope of a molecule present on the surface of an effector cell (i.e., an effector cell molecule ("ECM")), particularly a molecule present on the surface of a T lymphocyte, Natural Killer (NK) cell, or other monocyte. For example, such gp 41-binding molecules of the invention can be constructed to include a first epitope-binding domain that immunospecifically binds to an epitope of gp41 and a second epitope-binding domain that immunospecifically binds to an epitope of ECM, particularly CD2, CD3, CD8, CD16, T-cell receptor (TCR), or NKG 2D. In other embodiments of the invention, such binding molecules will additionally contain an epitope-binding domain sufficient to allow such molecules to bind to additional epitopes of the ECM and/or additional epitopes of molecules present on the surface of HIV-1 infected cells (e.g., a second epitope of gp41 or other epitopes of the HIV-1envelope protein). The invention also relates to pharmaceutical compositions comprising such molecules.

In one embodiment, such a molecule that binds gp41 will be bispecific but monovalent in order to have the ability to bind only a single epitope of gp41 and only a single epitope of a molecule present on the surface of an Effector Cell (ECM). Alternatively, such molecules may be multispecific and multivalent, i.e., capable of binding a total of one, two, three, or four epitopes, which may be assigned in any manner to bind one, two, or three epitopes of gp41 (where two or three gp41 epitopes may be the same or different) and three, two, or one epitope of one or more ECMs; or one or two epitopes of gp41 (wherein the two gp41 epitopes may be the same or different) and one or more two or one epitope of the ECM, and optionally one or two epitopes of one or more different HIV-1 molecules, particularly wherein such molecules are expressed on the surface of HIV-1 infected cells.

Thus, where such molecules are capable of immunospecifically binding to only a single ECM, they are capable of immunospecifically binding to:

(1) only one epitope of gp41 and one, two or three epitopes of the ECM (wherein two epitopes may be the same or different, and wherein three epitopes may be the same, or may be different, or may be two identical epitopes and one different epitope), or

(2) Only two epitopes of gp41 (where the two epitopes may be the same or different) and one or two epitopes of ECM (where the two epitopes may be the same or different); or

(3) Three gp41 epitopes (wherein the three epitopes may be the same, or may be different, or may be two identical epitopes and one different epitope) and an epitope of the ECM, or optionally they are capable of binding one or two gp41 epitopes (wherein the epitopes may be the same or different) and two or one epitope of a different HIV-1 molecule (wherein the two epitopes may be the same or different).

Similarly, where such a molecule is capable of immunospecifically binding to two different ECMs (e.g., a first ECM and a second ECM), they are capable of immunospecifically binding to:

(1) only one epitope of gp41 and one or two epitopes of a first ECM (where the two first ECM epitopes may be the same or different) and two or one epitope of a second ECM (where the two second ECMs may be the same or different), or

(2) Only two epitopes of gp41 (wherein the two epitopes may be the same or different) and one epitope of the first ECM and one epitope of the second ECM, or optionally they are capable of binding one epitope of gp41 and one epitope of a different HIV-1 molecule.

Non-limiting examples of such multispecific molecules capable of binding two epitopes are described below and include:

(1) DART-a, which binds to an epitope of gp41, and an epitope of ECM, CD 3; and

(2) DART-B, which binds to an epitope of gp41, and an epitope of ECM, CD 16.

Non-limiting examples of such multispecific molecules capable of binding three epitopes are described below and include:

(1) TRIDENT-A, which binds to one epitope of gp41, and to an epitope of two different ECMs (CD3 and CD 8);

(2) TRIDENT-B, which binds to an epitope of gp41, an epitope of ECM (CD3), and an epitope of a different HIV-1 molecule (gp 120);

(3) TRIDENT-C, which binds to an epitope of gp41, an epitope of ECM (CD16), and an epitope of a different HIV-1 molecule (gp 120); and

(4) TRIDENT-D, which binds two epitopes of gp41, and one epitope of ECM (CD 3).

Table 1 further illustrates possible combinatorial binding specificities of exemplary molecules of the invention.

By forming more complex molecules, molecules that bind gp41 that are capable of binding to one or more ECMs and optionally one or more different HIV-1 molecules with more than four epitope binding domains are available. Thus, the nature of the epitope and the further epitope that can be bound by the molecule of the invention is not limited as long as such further binding capability does not prevent such binding of a molecule or binding domain thereof capable of binding to an epitope of gp41 and does not prevent such binding of a molecule or binding domain thereof capable of binding to an epitope of the ECM. Thus, molecules of the invention that bind gp41 can be provided with alternative or additional epitope binding domains. As an example, the invention contemplates a molecule that binds gp41 that includes a first epitope binding domain capable of immunospecifically binding to an epitope of gp41 and a second epitope binding domain capable of immunospecifically binding to an epitope of ECM and a third epitope binding domain capable of immunospecifically binding to a different ECM or optionally a different HIV-1 molecule.

1. Diabodies that multispecific bind gp41 lacking an Fc domain

In one embodiment, the multispecific gp 41-binding molecule of the present invention will be a bispecific diabody and will include a domain capable of binding both the first epitope and the second epitope, but will lack an Fc domain and, therefore, will not be able to bind to an Fc γ R molecule via Fc-Fc γ R interactions. However, such molecules are capable of binding gp41 via the SDR or CDR of their gp41 binding domain. Thus, deletion of the Fc domain serves to prevent the molecule from binding to the Fc γ R, such as inhibiting the receptor CD 32B.

This type of bispecific diabodyThe first polypeptide chain of an embodiment preferably comprises in the N-terminal to C-terminal direction: n-terminal VL domain of a monoclonal antibody capable of binding a first epitope or a second epitope (i.e., VL)_gp41Or VL_{Second epitope}) A first intervening spacer peptide (linker 1), a VH domain of a monoclonal antibody capable of binding a second epitope (if this first polypeptide chain comprises a VL_gp41) Or a VH domain of a monoclonal antibody capable of binding gp41 (if this first polypeptide chain comprises a VL_{Second epitope}) A second intervening spacer peptide (linker 2) optionally containing a cysteine residue, a heterodimer promoting domain and a C-terminus (fig. 1A-1B).

The second polypeptide chain of this embodiment of the bispecific diabody comprises in the N-terminal to C-terminal direction: n-terminal VL domain of a monoclonal antibody capable of binding a first epitope or a second epitope (i.e., VL)_gp41Or VL_{Second epitope}And is a VL domain not selected for inclusion in the first polypeptide chain of the diabody), an intervening spacer peptide (linker 1), a VH domain of a monoclonal antibody capable of binding a first epitope or a second epitope (i.e., VH)_gp41Or VH_{Second epitope}And is a VH domain not selected for inclusion in the first polypeptide chain of the diabody), a second intervening spacer peptide (linker 2) optionally containing cysteine residues, a heterodimer promoting domain, and a C-terminus (fig. 1A-fig. 1B). The VL and VH domains employed that are specific for a particular epitope are preferably obtained from or derived from the same monoclonal antibody. However, such domains may be derived from different monoclonal antibodies, provided that they associate to form a functional binding domain capable of immunospecifically binding to such an epitope. Such different antibodies are referred to herein as "corresponding" antibodies.

The VL domain of the first polypeptide chain of such diabodies interacts with the VH domain of the second polypeptide chain of such diabodies to form a first functional epitope binding domain specific for one epitope (e.g., a first epitope or a second epitope). Similarly, the VL domain of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second epitope (i.e., the second epitope)Or a first epitope) specific second functional epitope binding domain. Thus, the selection of the VL and VH domains of the first and second polypeptide chains is "coordinated" such that the two polypeptide chains of the diabody collectively comprise a VL domain and a VH domain capable of binding both the first and second epitopes (i.e., they collectively comprise a VL)_{First epitope}/VH_{First epitope}And VL_{Second epitope}/VH_{Second epitope}) E.g. VL_gp41/VH_gp41And VL_{Second epitope}/VH_{Second epitope}。

Most preferably, the length of the intervening spacer peptide (i.e., "linker 1") separating such VL and VH domains is selected to substantially or completely prevent the VL and VH domains of the polypeptide chain from binding to each other (e.g., consisting of 0, 1, 2, 3,4, 5,6, 7,8, or 9 intervening linker amino acid residues). Thus, the VL domain and the VH domain of the first polypeptide chain are substantially or completely incapable of binding to each other. Similarly, the VL domain and the VH domain of the second polypeptide chain are substantially or completely incapable of binding to each other. Preferred intervening spacer peptides (linker 1) have the sequence (SEQ ID NO: 16): GGGSGGGG.

The length and composition of the second intervening spacer peptide ("linker 2") is selected based on the selection of one or more polypeptide domains that promote such dimerization (i.e., "heterodimer promoting domains"). Typically, the second intervening spacer peptide ("linker 2") will be between 3 and 20 amino acid residues in length. In particular, where a heterodimer-promoting domain is employed that does not include a cysteine residue, a second intervening cysteine-containing spacer peptide (linker 2) is utilized. The second intervening cysteine-containing spacer peptide (linker 2) will contain 1, 2, 3, or more than 3 cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence GGCGGG (SEQ ID NO: 17). Alternatively, linker 2 does not include cysteine (e.g., GGG, GGGS (SEQ ID NO:18), LGGGSG (SEQ ID NO:19), GGGSGGGSGGG (SEQ ID NO:20), ASTKG (SEQ ID NO:21), LEPKSS (SEQ ID NO:22), APSSS (SEQ ID NO:23), etc.) and uses a cysteine-containing heterodimer-promoting domain as described below. Optionally, both a cysteine-containing linker 2 and a cysteine-containing heterodimer promoting domain are used.

The heterodimer promoting domain may be GVEPKSC (SEQ ID NO:24) or VEPKSC (SEQ ID NO:25) or AEPKSC (SEQ ID NO:26) on one polypeptide chain and GFNRGEC (SEQ ID NO:27) or FNRGEC (SEQ ID NO:28) on the other polypeptide chain (US 2007/0004909).

In a preferred embodiment, the heterodimer promoting domain will comprise helical domains of tandem repeats of opposite charge, e.g., an "E-helix" heterodimer promoting domain (SEQ ID NO: 29:EVAALEK-EVAALEK-EVAALEK-EVAALEK) at pH 7 its glutamic acid residue will form a negative charge; or a "K-helix" heterodimer promoting domain (SEQ ID NO: 30:KVAALKE-KVAALKE-KVAALKE-KVAALKE) at pH 7, its lysine residue will form a positive charge. The presence of such charged domains facilitates association between the first and second polypeptides, and thus heterodimer formation. Modified heterodimer promoting domains comprising the above-described E-helix and K-helix sequences may be utilized so as to include one or more cysteine residues. The presence of such cysteine residues allows the covalent binding of a helix present on one polypeptide chain to a complementary helix present on another polypeptide chain, thereby covalently binding the polypeptide chains to each other and increasing the stability of the diabody. Such particularly preferred examples include those having an amino acid sequenceEVAACEK-EVAALEK-EVAALEK-EVAALEModified E-helices of K (SEQ ID NO:31), and having an amino acid sequenceKVAACKE-KVAALKE-KVAALKE-KVAALKThe heterodimer promoting domain of the modified K-helix of E (SEQ ID NO: 32).

As disclosed in WO 2012/018687, in order to improve the in vivo pharmacokinetic properties of the diabodies, the diabodies may be modified to comprise a polypeptide moiety at one or more ends of the diabodies that binds to a protein of serum. Most preferably, the polypeptide portion of such serum-binding protein will be placed at the C-terminus of the polypeptide chain of the diabody. Albumin is the most abundant protein in plasma and has a 19-day half-life in humans. Albumin is provided with several small molecule binding domains that allow albumin to bind non-covalently to other proteins, thereby extending their serum half-life. Albumin Binding domain 3(ABD3) Of protein G Of Streptococcus (Streptococcus) strain G148 consists Of 46 amino acid residues forming a stable triple helix bundle And has broad Specificity For Binding Albumin (Johansson, M.U. et al (2002) "Structure, Specificity, And model Of Interaction For Bacterial Albumin Binding Modules," J.biol.chem.277(10): 8114) 8120). Thus, a particularly preferred polypeptide portion of a serum-binding protein for improving the in vivo pharmacokinetic properties of diabodies is albumin-binding domain from streptococcal protein G (ABD), and more preferably albumin-binding domain 3 of protein G of streptococcal strain G148 (ABD3) (SEQ ID NO: 33): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP are provided.

As disclosed in WO 2012/162068 (incorporated herein by reference), the "deimmunized" variant of SEQ ID NO:33 has the ability to attenuate or eliminate MHC class II binding. Based on the combined mutation results, the following combination of substitutions are considered to be preferred substitutions for the formation of such de-immunized ABDs: 66D/70S + 71A; 66S/70S + 71A; 66S/70S + 79A; 64A/65A/71A; 64A/65A/71A + 66S; 64A/65A/71A + 66D; 64A/65A/71A + 66E; 64A/65A/79A + 66S; 64A/65A/79A + 66D; 64A/65A/79A + 66E. Variant ABD have modifications L64A, I65A and D79A or modifications N66S, T70S and D79A. Particularly preferably having the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNLID ₆₆NAKS ₇₀ A ₇₁EGVKALIDE ILAALP(SEQ ID NO:34)，

or the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNA ₆₄ A ₆₅NNAKT VEGVKALIA ₇₉E ILAALP(SEQ ID NO:35)，

or the amino acid sequence:

LAEAKVLANR ELDKYGVSDY YKNLIS ₆₆NAKS ₇₀ VEGVKALIA ₇₉a variant of E ILAALP (SEQ ID NO:36) deimmunized ABD, as this deimmunized ABD exhibits substantially wild-type binding while providing reduced MHC class II binding. Thus, the first polypeptide chain of such diabodies with ABD contains a third linker (linker 3), which is preferably positioned C-terminal to the E-helix (or K-helix) domain of such polypeptide chain so as to be interposed between the E-helix (or K-helix) domain and the ABD, which is preferably a de-immunized ABD. A preferred sequence for such linker 3 is SEQ ID NO 18: GGGS.

2. Multispecific gp 41-binding diabodies comprising an Fc domain

One embodiment of the invention relates to multispecific diabodies (e.g., bispecific, trispecific, tetraspecific, etc.) that include an Fc domain and are capable of simultaneously binding an epitope of gp41 and a second epitope (e.g., an epitope of a different HIV-1envelope protein, or an epitope expressed on the surface of an effector cell). The Fc domain of such molecules may be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG 4). The molecule may further comprise a CH1 domain and/or a hinge domain. When present, the CH1 domain and/or hinge domain can be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG4), and preferably is of the same isotype as the desired Fc domain.

The addition of IgG CH2-CH3 domains to one or both of the diabody polypeptide chains, such that diabody chain complexation results in the formation of Fc domains, increasing the biological half-life and/or altering the potency of the diabody. Such diabodies comprise two or more polypeptide chains having a sequence that allows the polypeptide chains to covalently bind to each other to form a covalently associated diabody that is capable of simultaneously binding gp41 and a second epitope. The incorporation of IgG CH2-CH3 domains onto both polypeptides of the diabody will allow the formation of a two-chain bispecific Fc domain-containing diabody (fig. 2).

Alternatively, the incorporation of IgG CH2-CH3 domains onto only one polypeptide of a diabody would allow the formation of more complex four-chain bispecific Fc domain-containing diabodies (fig. 3A-fig. 3C). FIG. 3C shows a representative four-chain with Constant Light (CL) and constant heavy CH1 domainsHowever, portions of such domains and other polypeptides may alternatively be employed (see, e.g., FIGS. 3A and 3B, U.S. patent publication Nos. 2013-0295121; 2010-0174053 and 2009-0060910; European patent publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT publication No. WO 2012/162068; WO 2012/018687; WO 2010/080538). Thus, for example, instead of the CH1 domain, a peptide having the amino acid sequence GVEPSSC (SEQ ID NO:24), VEPKSC (SEQ ID NO:25) or AEPKSC (SEQ ID NO:26) derived from the hinge domain of human IgG may be used, and instead of the CL domain, the C-terminal 6 amino acids GFNRGEC (SEQ ID NO:27) or FNRGEC (SEQ ID NO:28) of the human kappa light chain may be used. A four-chain diabody containing representative peptides is shown in fig. 3A. Alternatively, or in addition, tandem helical domains comprising opposite charges, such as "E-helix" helical domains (SEQ ID NO: 29:EVAALEK-EVAALEK-EVAALEK-EVAALEk or SEQ ID NO 31:EVAACEK-EVAALEK-EVAALEK-EVAALEK) (ii) a And a "K-helix" domain (SEQ ID NO: 30:KVAALKE-KVAALKE-KVAALKE-KVAALKe or SEQ ID NO: 32:KVAACKE-KVAALKE-KVAALKE-KVAALKE) the peptide of (1). In figure 3B shows a representative helical domain containing four chain double antibody.

The Fc domain-containing diabody molecules of the present invention may include additional intervening spacer peptides (linkers), typically such linkers will be incorporated between the heterodimer promotion domain (e.g., E-helix or K-helix) and the CH2-CH3 domain and/or between the CH2-CH3 domain and the variable domain (i.e., VH or VL). Typically, the additional linker will comprise 3-20 amino acid residues and may optionally contain all or a portion of the IgG hinge domain (preferably the cysteine-containing portion of the IgG hinge domain has 1, 2, 3 or more cysteine residues). Linkers that may be employed in the bispecific Fc domain-containing diabody molecules of the present invention include: GGGS (SEQ ID NO:18), LGGGSG (SEQ ID NO:19), GGGSGGGSGGG (SEQ ID NO:20), ASTKG (SEQ ID NO:21), LEPKSS (SEQ ID NO:22) APSSS (SEQ ID NO:23), APSSSPME (SEQ ID NO:37), VEPKSADKTHTCPPCP (SEQ ID NO:38), LEPKSADKTHTCPPCP (SEQ ID NO:39), DKTHTTPCPCP (SEQ ID NO: 40); a scFv linker: GGGGSGGGGSGGGGS (SEQ ID NO: 41); the "long" linker: GGGGSGGGSGGG (SEQ ID NO:42), GGC and GGG. For ease of cloning, the linker LEPKSS (SEQ ID NO:22) can be used in place of GGG or GGC. In addition, the peptide GGG or LEPKSS (SEQ ID NO:22) may be immediately followed by DKTHTTCPPCP (SEQ ID NO:40) to form an alternating linker: GGGDKTHTCPPCP (SEQ ID NO:43) and LEPKSSDKTHTCPPCP (SEQ ID NO: 44). In addition to or instead of a linker, the bispecific Fc domain-containing molecules of the invention may incorporate an IgG hinge domain. Exemplary hinge domains include: EPKSCDKTHTCPPCP from IgG1(SEQ ID NO:7), ERKCCVECPPCP from IgG2(SEQ ID NO:8), ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPP PCPRCPEPKSCDTPPPCPRCP from IgG3(SEQ ID NO:9), ESKYGPPCPSCP from IgG4(SEQ ID NO:10) and ESKYGPPCPPCP (SEQ ID NO:11), IgG4 hinge variant including a stable S228P substitution (shown underlined) (as numbered by the EU index shown in Kabat) to reduce chain exchange.

As provided in fig. 3A-3C, Fc domain-containing diabodies of the present invention can include four chains. The first and third polypeptide chains of this diabody contain three domains: (i) a VL 1-containing domain, (ii) a VH 2-containing domain, (iii) a heterodimer-promoting domain, and (iv) a CH2-CH 3-containing domain. The second and fourth polypeptide chains comprise: (i) a VL 2-containing domain, (ii) a VH 1-containing domain, and (iii) a heterodimer promotion domain, wherein the heterodimer promotion domain promotes dimerization of the first/third polypeptide chains with the second/fourth polypeptide chains. The VL and/or VH domains of the third and fourth polypeptide chains and the VL and/or VH domains of the first and second polypeptide chains may be the same or different so as to allow monospecific, bispecific or tetraspecific tetravalent binding. The symbols "VL 3" and "VH 3" represent the light chain variable domain and variable heavy chain domain, respectively, of such a diabody that binds a "third epitope". Similarly, the symbols "VL 4" and "VH 4" represent the light chain variable domain and variable heavy chain domain, respectively, of such a diabody that binds to a "fourth epitope". The overall structure of the polypeptide chains of representative four-chain bispecific Fc domain-containing diabodies of the present invention is provided in table 2:

HPD ═ heterodimer promoting domains

In a specific embodiment, the diabodies of the invention are bispecific, tetravalent (i.e., having four epitope binding structures), Fc-containing diabodies, which are composed of a total of four polypeptide chains (fig. 3A-3C). The bispecific, tetravalent, Fc-containing diabodies of the present invention comprise two first epitope-binding domains and two second epitope-binding domains.

In a further embodiment, the Fc domain-containing diabodies of the present invention can comprise three polypeptide chains. The first polypeptide of this diabody contains three domains: (i) a domain comprising VL1, (ii) a domain comprising VH2, and (iii) a domain comprising a CH2-CH3 sequence. The second polypeptide of such diabody comprises: (i) a VL 2-containing domain, (ii) a VH 1-containing domain, and (iii) a domain that promotes heterodimerization and covalent binding to the first polypeptide chain of a diabody. The third polypeptide of such diabodies includes the sequence CH2-CH 3. Thus, the first and second polypeptide chains of such diabodies associate together to form a VL1/VH1 epitope binding domain capable of binding to a first or second epitope, and a VL2/VH2 epitope binding domain capable of binding to another such epitope. The first and second polypeptides are bound to each other by a disulfide bond involving cysteine residues in their respective third domains. Note that the first and third polypeptide chains are complexed to each other via disulfide bonds to form a stable Fc domain. Such bispecific diabodies have enhanced efficacy. Fig. 4A and 4B illustrate the structure of such diabodies. Such Fc domain-containing diabodies can have one of two orientations (table 3):

HPD ═ heterodimer promoting domains

In a specific embodiment, the diabodies of the invention are bispecific, bivalent (i.e., having two epitope-binding domains), Fc-containing diabodies, which consist of a total of three polypeptide chains (fig. 4A-4B). The bispecific, bivalent Fc-containing diabodies of the invention comprise one epitope-binding domain that is immunospecific for a first epitope or a second epitope, and a VL2/VH2 epitope-binding domain that is capable of binding to another such epitope.

In further embodiments, the Fc domain-containing diabody can include a total of five polypeptide chains. In particular embodiments, two of the five polypeptide chains have the same amino acid sequence. The first polypeptide chain of such diabodies comprises: (i) a VH 1-containing domain, (ii) a CH 1-containing domain, and (iii) a CH2-CH 3-containing domain. The first polypeptide chain can be a heavy chain of an antibody comprising VH 1and a heavy chain constant region. The second and fifth polypeptide chains of such diabodies comprise: (i) a VL 1-containing domain and (ii) a CL-containing domain. The second polypeptide chain and/or the fifth polypeptide chain of such a diabody can be the light chain of an antibody comprising VL1 that is complementary to VH1 of the first/third polypeptide chains. The first polypeptide chain, the second polypeptide chain, and/or the fifth polypeptide chain can be isolated from a naturally occurring antibody. Alternatively, they may be constructed recombinantly. The third polypeptide chain of this diabody comprises: (i) a VH 1-containing domain, (ii) a CH 1-containing domain, (iii) a CH2-CH3 sequence-containing domain, (iv) a VL 2-containing domain, (v) a VH 3-containing domain, and (vi) a heterodimer-promoting domain, wherein the heterodimer-promoting domain promotes dimerization of a third chain and a fourth chain. The fourth polypeptide of this diabody contains: (i) a VL 3-containing domain, (ii) a VH 2-containing domain, and (iii) a domain that promotes heterodimerization and covalent binding to the third polypeptide chain of a diabody.

Thus, the first and second polypeptide chains and the third and fifth polypeptide chains of such a diabody associate together to form two VL1/VH1 epitope binding domains capable of binding a first epitope. The third and fourth polypeptide chains of such diabodies associate together to form a VL2/VH2 epitope binding domain capable of binding a second epitope, and a VL3/VH3 epitope binding domain capable of binding a third epitope. The first and third polypeptides are bound to each other by disulfide bonds involving cysteine residues in their respective constant regions. Note that the first and third polypeptide chains complex with each other to form an Fc domain. Such multispecific diabodies have enhanced efficacy. The structure of such diabodies is illustrated in figure 5. It will be appreciated that the VL1/VH1 domain, VL2/VH2 domain and VL3/VH3 domain may be the same or different, so as to allow monospecific, bispecific or trispecific binding.

The VL and VH domains of the polypeptide chain are selected so as to form a VL/VH epitope binding domain specific for the desired epitope. The VL/VH epitope binding domains formed by association of polypeptide chains can be the same or different so as to allow monospecific, bispecific, trispecific, or tetraspecific tetravalent binding. In particular, the VL domain and the VH domain may be selected such that the multivalent diabody may comprise two binding domains for a first epitope and two binding domains for a second epitope, or three binding domains for a first epitope and one binding domain for a second epitope, or two binding domains for a first epitope, one binding domain for a second epitope and one binding domain for a third epitope (as depicted in fig. 5). The overall structure of the polypeptide chains of a representative five-chain Fc domain-containing diabody of the present invention is provided in table 4:

HPD ═ heterodimer promoting domains

In a specific embodiment, the diabodies of the invention are bispecific, tetravalent (i.e., having four epitope-binding domains), Fc-containing diabodies, consisting of a total of five polypeptide chains, having two epitope-binding domains immunospecific for a first epitope and two epitope-binding domains specific for a second epitope. In another embodiment, the bispecific, tetravalent, Fc-containing diabodies of the invention comprise three epitope binding domains immunospecific for a first epitope and one epitope binding domain specific for a second epitope. As provided above, the VL domain and VH domain may be selected to allow tri-specific binding. Accordingly, trispecific, tetravalent, Fc-containing diabodies are also encompassed by the present invention. The trispecific, tetravalent, Fc-containing diabodies of the present invention comprise two epitope binding domains immunospecific for a first epitope, one epitope binding domain immunospecific for a second epitope and one epitope binding domain immunospecific for a third epitope.

In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a wide variety of responses ranging from effector functions such as antibody-dependent cellular cytotoxicity, large cell degranulation and phagocytosis to immunoregulatory signals such as regulation of lymphocyte proliferation and antibody secretion. All these interactions are initiated by the binding of the Fc domain of an antibody or immune complex to a cell surface receptor characteristic of hematopoietic cells. As discussed above, the diversity of cellular responses triggered by antibodies and immune complexes is dominated by three Fc receptors: the structural heterogeneity of Fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD16) arises. Fc γ RI (CD64), Fc γ RIIA (CD32A), and Fc γ RIII (CD16) are activating (i.e., immune system enhancing) receptors; fc γ RIIB (CD32B) is an inhibitory (i.e., immune system lowering) receptor. In addition, the interaction with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from endosomes to the cell surface and release to the blood. The amino acid sequences of exemplary wild-type IgG1(SEQ ID NO:12), IgG2(SEQ ID NO:13), IgG3(SEQ ID NO:14), and IgG4(SEQ ID NO:15) are presented above.

Modification of the Fc domain may result in altered phenotypes such as altered serum half-life, altered stability, altered sensitivity to cellular enzymes, or altered effector function. Therefore, it may be desirable to modify the Fc domain containing binding molecules of the invention with respect to effector function, for example, in order to enhance the efficacy of such molecules in the treatment of cancer. In certain instances, Fc domain mediated reduction or elimination of effector function is desirable, for example, where the mechanism of action of the antibody involves blocking or antagonism but does not kill cells bearing the target antigen. Increased effector function is often desired when directed against undesirable cells, such as tumors and foreign cells in which Fc γ Rs are expressed at low levels, e.g., tumor-specific B cells with low levels of Fc γ RIIB (e.g., non-hodgkin's lymphoma, CLL, and Burkitt's lymphoma). Molecules of the invention possessing such conferred or altered effector function activity are useful in the treatment and/or prevention of diseases, disorders (disorders) or infections in which enhanced efficacy of effector function activity is desired.

Accordingly, in certain embodiments, the Fc domain of the Fc domain-containing molecules of gp41 of the present invention can be an engineered variant Fc domain. While the Fc domain of the bispecific Fc domain-containing molecules of the invention may possess the ability to bind one or more Fc receptors (e.g., fcyr), more preferably such variant Fc domains have altered binding to fcyria (CD64), fcyriia (CD32A), fcyriib (CD32B), fcyriiia (CD16A), or fcyriiib (CD16B) (relative to the binding exhibited by a wild-type Fc domain), e.g., will have enhanced binding to activate a receptor and/or will have substantially reduced or no binding to inhibit a receptor. Thus, the Fc domain of the Fc domain-containing molecules of the invention may comprise part or all of the CH2 domain and/or part or all of the CH3 domain of the complete Fc domain, or may comprise a variant CH2 and/or variant CH3 sequence (which may comprise, for example, one or more insertions and/or one or more deletions relative to the CH2 domain or the CH3 domain of the complete Fc domain). Such Fc domains may include a non-Fc polypeptide portion, or may include a partially non-naturally intact Fc domain, or may include a non-naturally occurring directional CH2 domain and/or CH3 domain (such as, for example, two CH2 domains or two CH3 domains, or in an N-terminal to C-terminal direction, a CH3 domain linked to a CH2 domain, etc.).

Modifications Of the Fc domain identified as altering effector function are known In the art, including modifications that increase binding-Activating Receptors (e.g., Fc γ RIIA (CD16A) And decrease binding-inhibiting Receptors (e.g., Fc γ RIIB (CD32B) (see, e.g., Stavenhagen, J.B. et al (2007) "Fc Optimization Of Therapeutic Antibodies engineering disease To Kill Tumor Cells In Vitro And control molecules Expansion In Vila Via Low-Affinity activation Fcgamma Receptors," Cancer Res.57(18): 8882. about. 8890.) Table 5 lists exemplary modifications that increase binding-Activating Receptors And/or decrease binding-inhibiting Receptors for triple, double, quadruple or quintuple substitutions ((And numbering according To the EU index) relative To the amino acid sequence as shown above: ID 12)

Exemplary variants with reduced binding to CD32B and/or increased binding to human IgG1 Fc domain of CD16A contain F243L, R292P, Y300L, V305I, or P396L substitutions, where numbering is that of the EU index as in Kabat. These amino acid substitutions may be present in any combination in the human IgG1 Fc domain. In one embodiment, the variant human IgG1 Fc domain contains F243L, R292P, and Y300L substitutions. In another embodiment, the variant human IgG1 Fc domain contains F243L, R292P, Y300L, V305I, and P396L substitutions.

In certain embodiments, the Fc domain of the Fc domain-containing binding molecules of the present invention preferably exhibits reduced (or substantially NO) binding to Fc γ RIA (CD64), Fc γ RIIA (CD32A), Fc γ RIIB (CD32B), Fc γ RIIIA (CD16A), or Fc γ RIIIB (CD16B) (relative to the binding exhibited by wild-type IgG1 Fc domain (SEQ ID NO: 12)). In a specific embodiment, the Fc domain containing binding molecules of the invention comprise IgG Fc domains that exhibit reduced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. In preferred embodiments, the CH2-CH3 domain of such a binding molecule comprises any 1, 2, 3 or 4 of the following substitutions: L234A, L235A, D265A, N297Q and N297G, wherein the numbering is that of the EU index as in Kabat. In another embodiment, the CH2-CH3 domain contains an N297Q substitution, an N297G substitution, L234A and L235A substitutions, or a D265A substitution, as these mutations abolish FcR binding. Alternatively, the CH2-CH3 domain of a naturally occurring Fc domain that inherently exhibits reduced (or substantially NO) binding to Fc γ RIIIA (CD16A) and/or reduced effector function (relative to the binding and effector function exhibited by wild-type IgG1 Fc domain (SEQ ID NO: 12)) is utilized. In particular embodiments, the Fc domain-containing binding molecules of the present invention comprise an IgG2 Fc domain (SEQ ID NO:13), an IgG3 Fc domain (SEQ ID NO:14), or an IgG4 Fc domain (SEQ ID NO: 15). When utilizing an IgG4 Fc domain, the invention also contemplates the introduction of stable mutations, such as the hinge region S228P substitutions described above (see, e.g., SEQ ID NO: 11). Because the N297G, N297Q, L234A, L235A, and D265A substitutions eliminate effector functions, in situations where effector functions are desired, it may be preferable not to employ these substitutions.

Preferred IgG1 sequences of the CH2 and CH3 domains of the Fc domain-containing molecules of the invention having reduced or eliminated effector function will include substitutions L234A/L235A (SEQ ID NO: 45): APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGX

WhereinXIs lysine (K) or absent.

The serum half-life of a protein comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain for FcRn. The term "half-life" as used herein means the pharmacokinetic properties of a molecule, which is a measure of the average survival time of the molecule after its administration. Half-life may be expressed as the time required to eliminate fifty percent (50%) of a known amount of a molecule from the body of a subject (e.g., a human patient or other mammal) or a particular compartment thereof, e.g., as measured in serum, i.e., circulating half-life, or in other tissues. Generally, an increase in half-life results in an increase in the Mean Residence Time (MRT) of the administered molecule in the circulation.

In some embodiments, the Fc domain-containing binding molecules of the present invention comprise a variant Fc domain that includes at least one amino acid modification relative to a wild-type Fc domain such that the molecule has an increased half-life (relative to such a molecule if a wild-type Fc domain is included). In some embodiments, the Fc domain containing binding molecules of the present invention comprise a variant IgG Fc domain comprising half-life extending amino acid substitutions at one or more positions selected from the group consisting of: 238. 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435 and 436, wherein the numbering is that of the EU index as in Kabat. Many mutations that can increase the half-life of Fc domain containing molecules are known in the art and include, for example, M252Y, S254T, T256E, and combinations thereof. For example, see the mutations described in: U.S. Pat. nos. 6,277,375, 7,083,784, 7,217,797, 8,088,376; U.S. publication nos. 2002/0147311, 2007/0148164; and PCT publication nos. WO 98/23289, WO 2009/058492, and WO 2010/033279, which are incorporated herein by reference in their entirety.

In some embodiments, Fc domain-containing binding molecules of the invention that exhibit increased half-life have a variant Fc domain that includes substitutions at two or more of Fc domain residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435, and 436. In particular, the two or more substitutions are selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, a378V, M428L, N434A, H435K and Y436I, wherein the numbering is that of the EU index as in Kabat. In particular embodiments, such molecules may have a variant IgG Fc domain that includes the substitutions:

(A) M252Y, S254T and T256E;

(B) M252Y and S254T;

(C) M252Y and T256E;

(D) T250Q and M428L;

(E) T307Q and N434A;

(F) a378V and N434A;

(G) N434A and Y436I;

(H) V308P and N434A; or

(I) K288D and H435K.

In a preferred embodiment, the Fc domain containing molecules of gp41 of the present invention have a variant IgG Fc region that includes any 1, 2, or 3 of the following substitutions: M252Y, S254T and T256E. The invention further encompasses gp 41-binding molecules having a variant Fc region, comprising:

(A) one or more mutations that alter effector function and/or fcyr; and

(B) one or more mutations that increase serum half-life.

IgG1 sequences of the CH2 domain and CH3 domain of the Fc domain-containing molecules of the invention that provide increased half-life (and which have a 10-fold increase in Binding to both cynomolgus and Human FcRn) (Dall' Acqua, W.F. et al (2006) "Properties of Human IgG1S Engineered for Enhanced Binding to the New biological Fc Receptor (FcRn)," J.biol.chem.281(33):23514-23524) will include substitutions M252Y/S254T/T256E (SEQ ID NO: 46):

APELLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGX

wherein X is lysine (K) or absent.

The alternative IgG1 sequence of the CH2 domain and the CH3 domain of the Fc domain-containing molecule of the invention that combines the reduced or eliminated effector function provided by the substitution L234A/L235A and the increased serum half-life provided by the substitution M252Y/S254T/T256E is provided by SEQ ID NO: 47:

APEAAGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGX

whereinXIs lysine (K) or absent.

For certain antibodies, diabodies, and trivalent binding molecules in which Fc domain-containing polypeptide chains having different amino acid sequences are desired (e.g., in which the Fc domain-containing polypeptide chains are desired to be non-identical), it is desired to reduce or prevent homodimerization between CH2-CH3 domains of the same chain (e.g., between CH2-CH3 domains of two first polypeptide chains or two third polypeptide chains). The sequences of the CH2 and/or CH3 domains of such polypeptide chains need not be identical and are advantageously modified to facilitate heterodimeric complexation between the two polypeptide chains. For example, amino acid substitutions (preferably, substitutions with amino acids comprising bulky side groups forming "knobs" (e.g., tryptophan)) can be introduced into the CH2 or CH3 domains such that steric interference will prevent interaction with similarly mutated domains and will force pairing of the mutated domains with domains into which complementary or adaptive mutations have been engineered, i.e., "holes" (e.g., substitutions with glycine). Such a set of mutations may be engineered into any pair of polypeptides that includes a CH2-CH3 domain that forms an Fc domain to promote heterodimerization. Protein Engineering methods that support Heterodimerization, with respect to homodimerization, are well known in The art, particularly with respect to Engineering Of immunoglobulin-like molecules, And are encompassed herein (see, e.g., Ridgway et al (1996) "'Knobs-endo-places' Engineering Of Antibody CH3 Domains For Heavy Chain hybridization," Protein Engr.9:617-621, Atwell et al (1997) "Stable heterocycles From remodelling The Domain Interface Of A host microorganism utilization," J.Mol.biol.270: 26-35, And Xie et al (2005) "A New Format Of Bispecific antibodies: high efficiency hybridization, Expression 296," Cell J.95: each incorporated herein by reference).

A preferred knob is formed by modifying the IgG Fc domain to contain the modification T366W. Preferred sockets are formed by modifying the IgG Fc domain to contain modifications T366S, L368A and Y407V. To facilitate purification of the polypeptide chain homodimers carrying the mortar from the Fc domain containing molecules of the final bispecific heterodimers, the protein a binding domains of the polypeptide chains carrying the CH2 and CH3 domains of the mortar are preferably mutated by an amino acid substitution at position 435 (H435R). Thus, the polypeptide chain homodimers carrying the mortar will not bind protein a, whereas the bispecific heterodimers will retain their ability to bind protein a via the protein a binding domain. In an alternative embodiment, the polypeptide chain carrying the mortar may incorporate amino acid substitutions at positions 434 and 435 (N434A/N435K).

The preferred IgG1 amino acid sequence of the CH2 domain and CH3 domain of one Fc domain-containing polypeptide chain of the Fc domain-containing molecules of the invention will have a "knob-carrying" sequence (SEQ ID NO: 48):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGX

whereinXIs lysine (K) or absent.

Alternative IgG1 amino acid sequences for the CH2 and CH3 domains of one Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention having the M252Y/S254T/T256E substitution and the sequence "carrying the knob" are SEQ ID NO: 49:

APEAAGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGX

wherein X is lysine (K) or absent.

A preferred IgG1 amino acid sequence of the CH2 domain and CH3 domain of another Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention having two polypeptide chains (or a third polypeptide chain of an Fc domain-containing molecule having three, four or five polypeptide chains) will have a sequence "carrying a mortar" (SEQ ID NO: 50):

APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQGNVFSCSVMHE ALHNRYTQKS LSLSPGX

whereinXIs lysine (K) or absent.

Alternative IgG1 amino acid sequences for the CH2 and CH3 domains of the other Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention having the M252Y/S254T/T256E substitution and the sequence "carrying the mortar" are SEQ ID NO: 51:

APEAAGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVDGVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQGNVFSCSVMHE ALHNRYTQKS LSLSPGX

whereinXIs lysine (K) or absent.

The IgG4 amino acid sequence of the CH2 and CH3 domains of one Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention has an increased serum half-life (relative to the IgG1 CH2 and CH3 domains) because it possesses the Y252/T254/E256(SEQ ID NO: 52):

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVDGVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGX

whereinXIs lysine (K) or absent.

This "knob carrying" variant of the IgG4 CH2-CH3 amino acid sequence has the amino acid sequence of SEQ ID NO: 53:

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVDGVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLWCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGX

whereinXIs lysine (K) or absent.

This "mortar carrying" variant of the IgG4 CH2-CH3 amino acid sequence has the amino acid sequence of SEQ ID NO: 54:

APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVDGVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLSCAVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSRL TVDKSRWQEGNVFSCSVMHE ALHNRYTQKS LSLSLGX

whereinXIs lysine (K) or absent.

As will be noted, the CH2-CH3 domains of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54 include a substitution with alanine at position 234 and alanine at position 235, and thus form Fc domains that exhibit reduced (or substantially NO) binding to Fc γ RIA (CD64), Fc γ RIIA (CD32A), Fc γ RIIB (CD32B), Fc γ RIIIA (CD16A) or Fc γ RIIIB (CD16B) (relative to the binding exhibited by the wild-type Fc domain (SEQ ID NO: 12)). The invention also encompasses such CH2-CH3 domains, which include wild-type alanine residues, alternative substitutions and/or additional substitutions that modify the effector function and/or F γ R binding activity of the Fc domain. The present invention also encompasses such CH2-CH3 domains that further include one or more half-life extending amino acid substitutions. In particular, the invention encompasses such hole-carrying and such knob-carrying CH2-CH3 domains further comprising M252Y/S254T/T256E.

Preferably, the first polypeptide chain will have a "knob-carrying" CH2-CH3 sequence, such as that of SEQ ID NO:48 or SEQ ID NO: 49. However, as will be appreciated, the CH2-CH3 domain (e.g., SEQ ID NO:50 or SEQ ID NO:51) that "carries a hole" may be employed in the first polypeptide chain, in which case the CH2-CH3 domain (e.g., SEQ ID NO:48 or SEQ ID NO:49) that "carries a knob" would be employed in the second polypeptide chain of the Fc domain-containing molecule of the invention having two polypeptide chains (or in the third polypeptide chain of the Fc domain-containing molecule having three, four or five polypeptide chains).

In other embodiments, the invention encompasses Fc domain-containing binding molecules comprising a CH2 domain and/or a CH3 domain that have been engineered to favor heterodimerization over homodimerization using mutations known in the art, such as those disclosed in PCT publication nos. WO 2007/110205, WO 2011/143545, WO 2012/058768, WO 2013/06867, WO 2014/081955, WO 2016/086189, all of which are incorporated herein by reference in their entireties.

3. Trivalent binding molecules containing Fc domains

A further embodiment of the invention relates to a trivalent binding molecule comprising an Fc domain and capable of simultaneously binding a first epitope, a second epitope and a third epitope, wherein at least one of these epitopes is not identical to another epitope. Such trivalent binding molecules include three epitope binding domains, two of which are diabody-type binding domains that provide binding domain a and binding domain B, and one of which is a Fab-type binding domain or an scFv-type binding domain that provides binding domain C (see, e.g., fig. 6A-6F, PCT publications WO 2015/184207 and WO 2015/184203). Such trivalent binding molecules thus include the "VL 1"/"VH 1" domain of such trivalent binding molecules capable of binding a first epitope and the "VL 2"/"VH 2" domain capable of binding a second epitope and the "VL 3" and "VH 3" domains capable of binding a third epitope. A "diabody-type binding domain" is a type of epitope binding domain present in a diabody, as described above, in which a single polypeptide chain includes both a VL domain and a VH domain, but such domains do not interact to form an epitope binding site. Each of the "Fab-type binding domain" and "scFv-type binding domain" is of the type of epitope binding domain present in an antibody, as described above, wherein a single polypeptide chain comprises (1) a VL domain or a VH domain, and a second polypeptide chain comprises the corresponding VH domain or VL domain, so as to form an epitope binding site, or (2) both a VL domain and a VH domain, wherein such domains interact to form an epitope binding site. Fab-type binding domains also differ from diabody-type binding domains in that the two polypeptide chains forming the Fab-type binding domain comprise only a single epitope binding domain, whereas the two polypeptide chains forming the diabody-type binding domain comprise at least two epitope binding domains. Similarly, scFv-type binding domains differ from diabody-type binding domains in that they comprise only a single epitope-binding domain. Thus, Fab-type and scFv-type binding domains as used herein are distinct from diabody-type binding domains.

Typically, a molecule of the invention that binds trivalent gp41 will comprise four different polypeptide chains (see fig. 6A-6B), however, a molecule can comprise a fewer or greater number of polypeptide chains, for example, by fusing such polypeptide chains to each other (e.g., via peptide bonds), or by splitting such polypeptide chains to form additional polypeptide chains, or by associating fewer or additional polypeptide chains via disulfide bonds. Fig. 6C-6F illustrate this aspect of the invention by schematically depicting such a molecule with three polypeptide chains. As provided in fig. 6A-6F, trivalent binding molecules of the invention may have alternative orientations in which the diabody-type binding domains are N-terminal (fig. 6A, 6C, and 6D) or C-terminal (fig. 6B, 6E, and 6F) to the Fc domain. The CH2 and CH3 domains useful for generating trivalent binding molecules are provided above and include a knob-carrying and a hole-carrying domain.

In certain embodiments, the first polypeptide chain of such trivalent gp 41-binding molecules of the invention comprises: (i) a VL 1-containing domain, (ii) a VH 2-containing domain, (iii) a heterodimer-promoting domain, and (iv) a CH2-CH 3-containing domain. The VL1 domain and VL2 domain are located N-terminal or C-terminal to the CH2-CH 3-containing domain, as shown in table 5 (see also fig. 6A and 6B). The second polypeptide chain of this embodiment comprises: (i) a VL 2-containing domain, (ii) a VH 1-containing domain, and (iii) a heterodimer-promoting domain. The third polypeptide chain of this embodiment comprises: (i) a VH 3-containing domain, (ii) a CH 1-containing domain, and (iii) a CH2-CH 3-containing domain. The third polypeptide chain can be a heavy chain of an antibody comprising VH3 and a heavy chain constant region or a polypeptide comprising such a domain. The fourth polypeptide of this embodiment comprises: (i) a VL 3-containing domain and (ii) a CL-containing domain. The fourth polypeptide chain can be an antibody comprising VL3 complementary to VH3 of the third polypeptide chain or a light chain of a polypeptide comprising such a domain. The third polypeptide chain or the fourth polypeptide chain can be isolated from a naturally occurring antibody. Alternatively, they may be constructed recombinantly, synthetically, or by other means.

The light chain variable domains of the first and second polypeptide chains are separated from the heavy chain variable domains of such polypeptide chains by an intervening spacer peptide having a length that is too short to allow their VL1/VH2 (or their VL2/VH1) domains to associate together to form an epitope binding domain capable of binding a first epitope or a second epitope. Preferred intervening spacer peptides (linker 1) for this purpose have the sequence (SEQ ID NO: 16): GGGSGGGG. The other domains of the trivalent binding molecule may be separated by one or more intervening spacer peptides (linkers) optionally including cysteine residues. In particular, as provided above, such linkers will typically be incorporated between the variable domain (i.e., VH or VL) and the peptide heterodimer promoting domain (e.g., E-helix or K-helix) as well as between such peptide heterodimer promoting domain (e.g., E-helix or K-helix) and the CH2-CH3 domain. Exemplary linkers for generating trivalent binding molecules are provided above and are also provided in international patent publication nos.: WO 2015/184207 and WO 2015/184203. Thus, the first and second polypeptide chains of such a trivalent binding molecule associate together to form a VL1/VH1 binding domain capable of binding a first epitope, and a VL2/VH2 binding domain capable of binding a second epitope. The third and fourth polypeptide chains of such a trivalent binding molecule associate together to form a VL3/VH3 binding domain capable of binding a third epitope.

As described above, a trivalent gp 41-binding molecule of the invention can include three polypeptides. A trivalent binding molecule comprising three polypeptide chains may be obtained by linking the domain of the fourth polypeptide at the N-terminus to the domain comprising VH3 of the third polypeptide (e.g. using an intervening spacer peptide (linker 4)). Alternatively, a third polypeptide chain of the trivalent binding molecule of the invention is utilized that contains the following domains: (i) a VL 3-containing domain, (ii) a VH 3-containing domain, and (iii) a CH2-CH 3-containing domain, wherein VL3 and VH3 are spaced apart from each other by an intervening spacer peptide that is sufficiently long (at least 9 or more amino acid residues) to allow these domains to associate to form an epitope-binding domain. One preferred intervening spacer peptide for this purpose has the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 41).

It will be appreciated that the VL1/VH1, VL2/VH2 and VL3/VH3 domains of such trivalent binding molecules may be different so as to allow monospecific, bispecific or trispecific binding. In particular, the VL domain and the VH domain may be selected such that the trivalent binding molecule comprises two binding domains for a first epitope and one binding domain for a second epitope, or one binding domain for a first epitope and two binding domains for a second epitope, or one binding domain for a first epitope, one binding domain for a second epitope and one binding domain for a third epitope.

The overall structure of the polypeptide chains of representative trivalent binding molecules of the invention is provided in FIGS. 6A-6H for neutralization

In table 6:

HPD ═ heterodimer promoting domains

As provided above, such trivalent binding molecules can include three, four, five, or more polypeptide chains.

Exemplary gp 41-binding molecules of the invention

The present invention relates to molecules (e.g., antibodies, diabodies, scFvs, antibodies) that bind to an epitope of gp41 HIV-1envelope (Env) protein that binds gp41,trident^TMEtc.) because they possess an optimized gp41 binding domain with the binding specificity of the 7B2 antibody.

Envelope (Env) glycoproteins of HIV

The envelope (Env) glycoprotein of HIV is expressed on the surface of prolific infected cells. HIV Type 1(HIV-1) enters the host Through all transmitting Mucosa in a process called Transcytosis (Shen, R. et al (2010) "GP 41-Specific Antibody Blocks Cell-Free HIV Type 1Transcytosis Through high Human Rectal Mucosa And Model colloid episum," J.Immunol.184(7): 3648-3655). It has been reported that HIV-1 Transcytosis Across the intestinal And genital epithelium involves viral components including Gp41 env Protein (Bomsel, M. et al (1998) "endothelial secretion Of HIV transduction Across light sources DNA Or IgM," Immunity 9: 277. 287; Alfsen, A. et al (2002) "HIV-1 Gp41 Envelope reagents 650. 685 amplified non Natural Virus Act A Protein To A library To reaction promoter reaction center," J. biol. chem. 25649. 25659; Alfsen, A. et al (2001) "polypeptide specificity A Protein antigen Protein Gp 32. Gp. III receptor Protein antigen binding Protein antigen Gp 1. G. Biogene antigen binding Protein, "Mucosal Immunol.2:412-426), gp120 and gp 160; and host epithelial Cell receptors and attachment molecules including glycosphingolipidgalactosylceramide, the co-receptors CCR5 and heparan sulfate proteoglycan attachment receptor, syndecans and aggregatins (Shen, R. et al (2010) "GP 41-Specific Antibody Blocks Cell-Free HIV Type 1 transduction polypeptides Through Human Recal Mucosa and Model collagen epitope Epithelium," J.Immunol.184(7): 3648) -3655.

Thus, gp41 can be used as a target for a cytotoxic Immunoconjugate (IC) In which a cell-killing moiety comprising a toxin, drug, or radionuclide is chemically or genetically linked to a molecule that binds gp41 (see, e.g., Pincus, S.H. et al (2017) "Design and In Vivo catalysis of Immunoconjugates Targeting HIV gp160," J.Virol.91(3). pi: e01360-16. doi: 10.1128/JVI.01360-16).

B. Antibody 7B2

Antibody 7B2(Genbank accession Nos. JX188438 and JX188439) is an anti-HIV env human IgG1 antibody that binds HIV gp41 at 598-604 in the immunodominant helix-loop-helix region Of the molecule (Sadraean, M. et al (2017) 'Selective cytotoxin Of A Novel Immunotoxin Based On one Pulchellin A Chain For Cells Expressing HIV Envelope,' Sci. Rep.7(1):7579 doi: 10.1038/s 418-017 5937-593). Antibodies were isolated from HIV-1 chronically infected subjects using Epstein-Barr (EB) viral B cell transformation and xenohybridoma production (Pincus, S.H. et al (2003) "In Vivo Efficacy Of Anti-Glycopterin 41, But Not-Glycopterin 120, immunoxins In A Mouse Model Of HIV Infection," J.Immunol.170(4): 2236-2241). It has been found that Antibody 7B2 is capable of recognizing both viral particles and infected cells (Santa, S. et al (2015) "Human Non-hybridizing HIV-1 endogenous Antibodies Limit the Number of Foundation Virus Infection in Rhesus Macaq," ploS Patholog.11 (8): e1005042. doi: 10.1371/joint. pant.1005042; Tay, M.Z. et al (2016) "Antibody-Mediated interaction of Infection HIV-1 virus Infection immune Infection Antibodies and Subcoses," PL Patholog.12 (8): 395817. doi: 10.1371/joint. 10017).

The amino acid sequence of the VL domain of the 7B2 antibody (SEQ ID NO:55) is shown below and in FIG. 7A (CDR)_LResidues are underlined):

DIVMTQSPDS LAVSPGERAT IHCKSSQTLL YSSNNRHSIA WYQQRPGQPPKLLLYWASMR LSGVPDRFSG SGSGTDFTLT INNLQAEDVA IYYCHQYSSHPPTFGHGTRV EIK

7B2 antiThe amino acid sequence of the VH domain of the body (SEQ ID NO:56) is shown below and in FIG. 7B (CDR)_HResidues are underlined):

QVQLVQSGGG VFKPGGSLRL SCEASGFTFT EYYMTWVRQAPGKGLEWLAY ISKNGEYSKY SPSSNGRFTI SRDNAKNSVF LQLDRLSADDTAVYYCARAD GLTYFSELLQ YIFDLWGQGA RVTVSS

C. optimized 7B2GL

The amino acid sequence of the VL domain of the optimized 7B2GL antibody (SEQ ID NO:57) is shown below and in FIG. 7A (CDR)_LResidues are underlined):

DIVMTQSPDS LAVSLGERAT INCKSSQTLL YSSNNRHSIA WYQQKPGQPP KLLIYWASMR LSGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCHQYSSH PPTFGQGTKV EIK

the amino acid sequence of the VH domain of the optimized 7B2GL antibody (SEQ ID NO:58) is shown below and in FIG. 7B (CDR)_HResidues are underlined):

QVQLVESGGG LVKPGGSLRL SCAASGFTFS EYYMTWIRQA PGKGLEWVSY ISKNGEYSKY SPSSNGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARAD GLTYFSELLQ YIFDLWGQGT LVTVSS

exemplary Effector cell binding Capacity

As provided herein, the present invention relates to a multispecific gp 41-binding molecule capable of binding to an epitope of gp41 and an epitope of an effector cell surface molecule ("ECM"). As used herein, the term "effector cell" refers to a cell that directly or indirectly mediates killing of a target cell (e.g., an exogenous cell, an infected cell, or a cancer cell). Examples of effector cells include helper T cells, cytotoxic T cells, Natural Killer (NK) cells, plasma cells (antibody-secreting B cells), macrophages, and granulocytes. Preferred effector cell surface molecules ("ECM") include CD2, CD3, CD8, CD16, TCR, and NKG2D receptors. Accordingly, molecules capable of immunospecifically binding to an epitope of such a molecule, or other effector cell surface molecule, can be used in accordance with the principles of the present invention. Exemplary antibodies, VH and/or VL domains thereof, and/or CDRs of the VL region are provided below_L1, 2 or all 3 ofAnd/or the CDRs of the VH domains_H1, 2 or all 3, can be used to construct molecules capable of mediating redirected killing of target cells.

A. Exemplary CD2 binding Capacity

In one embodiment, the molecule of the invention is capable of binding to an epitope of CD2 present on the surface of such effector cells. CD2 is a cell adhesion molecule found on the surface of T-cells and Natural Killer (NK) cells. CD2 enhances NK Cell Cytotoxicity, probably as a promoter of NK Cell Nanotube Formation (Mace, E.M. et al (2014) "Cell Biological Steps and Checkpoints in Accessing NK Cell Cytotoxicity," immunological Cell. biol.92(3): 245-255; Comerci, C.J. et al (2012) "CD 2 proteins Human Natural Killer Cell Membrane Nanotube Formation," PLoS One7(10): e47664: 1-12). Molecules that specifically bind to CD2 include the anti-CD2 antibody "CD 2 mAb Lo-CD2 a".

The amino acid sequence of the VH domain of CD2 mAb Lo-CD2a (ATCC accession No. HB-11423); SEQ ID NO:59) shown below (CDR)_HResidues are underlined): EVQLQQSGPE LQRPGASVKL SCKASGYIFTEYYMYWVKQR PKQGLELVGRIDPEDGSIDY VEKFKKKATL TADTSSNTAY MQLSSLTSED TATYFCARGKFNYRFAYWGQ GTLVTVSS

The amino acid sequence of the VL domain of CD2 mAb Lo-CD2a (ATCC accession No. 11423; SEQ ID NO:60) is shown below (CDR)_LResidues are underlined):

DVVLTQTPPT LLATIGQSVS ISCRSSQSLL HSSGNTYLNW LLQRTGQSPQPLIYLVSKLE SGVPNRFSGS GSGTDFTLKI SGVEAEDLGV YYCMQFTHYPYTFGAGTKLE LK

B. exemplary CD3 binding Capacity

In one embodiment, the molecule of the invention is capable of binding an epitope of CD 3. CD3 is a T-Cell co-Receptor consisting Of four distinct strands (Wucherpfennig, K.W. et al (2010) "Structural Biology Of The T-Cell Receptor: instruments Into Receptor Assembly, Ligand Recognition, And identification Of signalling," Cold Spring Harb. Perspectrum. biol.2(4): a 005140; pages 1-14). In mammals, the complex contains a CD3 γ chain, a CD3 δ chain, and two CD3 epsilon chains. These chains associate with molecules called T-cell receptors (TCRs) to generate activation signals in T lymphocytes. In the absence of CD3, the TCR is not suitable for assembly and is decomposed (Thomas, S. et al (2010) "Molecular Immunology threads From Therapeutic T-Cell Receptor Gene Transfer," Immunology 129(2): 170-. CD3 was found To bind To The membrane Of all mature T-cells And was found To be scarcely found In other Cell types (see Janeway, C.A. et al (2005) In: immunology: The Immune System In Health Disease, "6 th Of Garland Science Publishing, NY, pp.214-216; Sun, Z.J. et al (2001)" Mechanisms connecting To T Cell Receptor signalling And assisted recovering By The Solution Structure Of The CD 3. gamma. heterodorimer, "105 (7):913-," Kuhns, M.S. et al (nonconstracturing Of The membrane Function/Function Of The TCR 3; Febnix.139) And "Febnig.139). Molecules that specifically bind CD3 include the anti-CD3 antibodies "CD 3mAb 1" and "OKT 3". The anti-CD3 antibody CD3mAb1 is capable of binding to a non-human primate (e.g., cynomolgus monkey).

The amino acid sequence (SEQ ID NO:61) of the VH domain of humanized CD3mAb1 is shown below (CDR NO:61)_HResidues are underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

the amino acid sequence (SEQ ID NO:62) of the VL domain of humanized CD3mAb1 is shown below (CDR NO:62)_LResidues are underlined):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

the humanized variant "CD 3mAb1 (D65G)" may be incorporated. CD3mAb 1(D65G) includes the VL domain of CD3mAb 1(SEQ ID NO:62) and the VH CD3mAb1 domain with a D65G substitution (Kabat position 65, corresponding to residue 68 of SEQ ID NO: 63).

The amino acid sequence (SEQ ID NO:63) of the VH of CD3mAb 1(D65G) is shown inFollowing (CDR)_HResidues are underlined and the position of the substitution (D65G) is double underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

alternatively, humanized affinity variants of CD3mAb1 may be incorporated. Variants include low affinity variants designated "CD 3mAb1 low" and variants with faster off-rates (off rates) designated "CD 3mAb1 fast". The VL domain of CD mAb 1(SEQ ID NO:62) was universal for CD3mAb1 low and CD3mAb1 fast and is provided above. The amino acid sequences of the VH domains of each of CD3mAb1 low and CD3mAb1 fast are provided below.

The amino acid sequence of the low variable heavy chain domain of anti-human CD3mAb 1(SEQ ID NO:64) is shown below (CDR)_HResidues are underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVT WFAYWGQGTL VTVSS

the amino acid sequence of the variable heavy chain domain of anti-human CD3mAb 1(SEQ ID NO:65) is shown below (CDR)_HResidues are underlined):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HKNFGNSYVT WFAYWGQGTL VTVSS

another anti-CD3 antibody that may be utilized is the antibody Moluomab-CD 3 "OKT 3". (Xu et al (2000) "In Vitro Characterisation Of Five treated OKT3 Effect Function variants," cell. Immunol.200: 16-26); norman, D.J, (1995) "Mechanisms Of Action And Overview Of OKT3," the. drug Unit.17 (6): 615-; canamax, D.M. et al (1987) "Monoclonal Antibody (OKT3) Treatment Of Acute recovery Allograft project," Pharmacotherapy 7(4): 121-124; swinnen, L.J. et al (1993) "OKT 3 Monoclonal Antibodies index Interleukin-6And d Interleukin-10: a Passive Cable Of porous Disorders Associated With transfer, "curr. Opin. Nephrol. hypertens.2(4): 670-.

The amino acid sequence of the VH domain of OKT 3(SEQ ID NO:66) is shown below (CDR)_HResidues are underlined):

QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSS

the amino acid sequence of the VL domain of OKT 3(SEQ ID NO:67) is shown below (CDR)_LResidues are underlined):

QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDTSKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSGTKLEINR

additional anti-CD3 antibodies that may be utilized include, but are not limited to, those described in PCT publication nos. WO 2008/119566 and WO 2005/118635.

C. Exemplary CD8 binding Capacity

In one embodiment, the molecule of the invention is capable of binding to an epitope of CD8 present on the surface of such effector cells. CD8 is a T-cell co-receptor consisting of two distinct chains expressed on cytotoxic T-cells (Leahy, D.J. (1995) "A Structural View of CD4 and CD8," FASEB J., 9: 17-25). It has been found that by antigen: a major histocompatibility class I (MHC I) molecule complex arranged on the surface of a target cell and a peptide sequence of CD8⁺Co-stimulatory interactions between complexes of CD8 and T-cell receptors arrayed on the surface of T-cells mediate CD8⁺Activation of T-cells (Gao, G. and Jakobsen, B., (2000)' Molecular interactions of Receptor CD8 and MHC class I: the Molecular basis for functional coding with the T-Cell Receptor. Immunol Today 21: 630. 636). Unlike MHC II molecules, which are expressed only by certain cells of the immune system, MHC I molecules are very widely expressed. Thus, cytotoxic T cells are able to bind to a variety of cell types. Activated cytotoxic T cells mediate release of their cytotoxic perforins, granzymes and granulysinsAnd (4) killing the cells. Antibodies that specifically bind CD8 include the anti-CD 8 antibodies "OKT 8" and "TRX 2".

The amino acid sequence of the VH domain of OKT8 (SEQ ID NO:68) is shown below (CDR)_HResidues are underlined):

QVQLLESGPE LLKPGASVKM SCKASGYTFT DYNMHWVKQS HGKSLEWIGY IYPYTGGTGY NQKFKNKATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

the amino acid sequence of the VL domain of OKT8 (SEQ ID NO:69) is shown below (CDR)_LResidues are underlined):

DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMHWY QQKPGQPPKVLIYLASNLESGVPARFSGSG SRTDFTLTID PVEADDAATY YCQQNNEDPYTFGGGTKLEI KR

the amino acid sequence (SEQ ID NO:70) of the VH domain of TRX2 is shown below (CDR)_HResidues are underlined):

QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQA PGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKPHYDGYYHFFDS WGQGTLVTVS S

the amino acid sequence (SEQ ID NO:71) of the VL domain of TRX2 is shown below (CDR)_LResidues are underlined):

DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYN TDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQG TKVEIK

D. exemplary CD16 binding Capacity

In one embodiment, the molecule of the invention is capable of binding an epitope of CD 16. CD16 is expressed by Human IgG binding to aggregate but not monomeric Human IgG biological Neutrophils, eosinophils, Natural Killer (NK) Cells And tissue macrophages (Peltz, G.A. et al (1989) "Human Fc Gamma RIII: Cloning, Expression, And Identification Of The Chromosomal Cells Of Two FcRCepers Format IgG," Proc.Natl.Acad.Sci. (U.S.A.)86(3): 1013-1017; Bachanova, V.et al (2014) "NK Cells In Therapy Of Cells, Crit.Rev.Oncog.19(1-2): 141; Mill, J.S. (2013)" polypeptides: Natural kinase In Therapy cell III Cells, III. 12; protein kinase, III. 12. P.11-2) protein III: (protein 2) protein III-2, protein kinase, protein III-2, protein III, protein, "biochem. Soc. trans.30(4):507- & 511). Molecules that specifically bind CD16 include anti-CD 16 antibodies "3G 8" and "a 9". Humanized 3G8 ("h 3G 8") antibodies are described in PCT publication No. WO 03/101485.

The amino acid sequence of the VH domain of murine 3G8 (SEQ ID NO:72) is shown below (CDR)_HResidues are underlined):

QVTLKESGPG ILQPSQTLSL TCSFSGFSLR TSGMGVGWIR QPSGKGLEWL AHIWWDDDKR YNPALKSRLT ISKDTSSNQV FLKIASVDTA DTATYYCAQI NPAWFAYWGQ GTLVTVSA

the amino acid sequence of the VL domain of murine 3G8 (SEQ ID NO:73) is shown below (CDR)_LResidues are underlined):

DTVLTQSPAS LAVSLGQRAT ISCKASQSVD FDGDSFMNWY QQKPGQPPKLLIYTTSNLESGIPARFSASG SGTDFTLNIH PVEEEDTATY YCQQSNEDPYTFGGGTKLEI K

the amino acid sequence of the VH domain of h3G8 (SEQ ID NO:74) is shown below (CDR)_HResidues are underlined):

QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMGVGWIR QPPGKALEWLAHIWWDDDKR YNPALKSRLT ISKDTSKNQV VLTMTNMDPVDTATYYCAQI NPAWFAYWGQ GTLVTVSS

the amino acid sequence of the VL domain of murine h3G8 (SEQ ID NO:75) is shown below (CDR)_LResidues are underlined):

DIVMTQSPDS LAVSLGERAT INCKASQSVD FDGDSFMNWYQQKPGQPPKL LIYTTSNLESGVPDRFSGSG SGTDFTLTIS SLQAEDVAVYYCQQSNEDPY TFGQGTKLEI K

the amino acid sequence of the VH domain of A9 (SEQ ID NO:76) is shown below (CDR)_HResidues are underlined)：

QVQLQQSGAE LVRPGTSVKI SCKASGYTFT NYWLGWVKQR PGHGLEWIGD IYPGGGYTNY NEKFKGKATV TADTSSRTAY VQVRSLTSED SAVYFCARSASWYFDVWGAR TTVTVSS

The amino acid sequence of the VL domain of A9 (SEQ ID NO:77) is shown below (CDR)_LResidues are underlined):

DIQAVVTQES ALTTSPGETV TLTCRSNTGT VTTSNYANWV QEKPDHLFTGLIGHTNNRAPGVPARFSGSL IGDKAALTIT GAQTEDEAIY FCALWYNNHWVFGGGTKLTVL

U.S. patent No. 9,035,026 describes anti-CD 16 antibodies that are capable of binding CD16A but not CD 16B. Such antibodies are particularly useful as components of bispecific or multispecific binding molecules directed against disease-associated cells, as they primarily recruit NK cells and are not bound by circulating soluble CD16B or translocated from NK cell binding by binding to neutrophils or activated eosinophils.

The amino acid sequence (SEQ ID NO:78) of the VH domain of the anti-CD 16A antibody of U.S. Pat. No. 9,035,026 is shown below (CDR)_HResidues are underlined):

EVQLVQSGAE VKKPGESLKV SCKASGYTFT SYYMHWVRQAPGQGLEWMGI INPSGGSTSY AQKFQGRVTM TRDTSTSTVY MELSSLRSEDTAVYYCARGS AYYYDFADYW GQGTLVTVSS

the amino acid sequences of seven suitable VL domains of this anti-CD 16A antibody of U.S. Pat. No. 9,035,026 are shown below as SEQ ID NOS: 79-85:

SYELMQPPSV SVSSGQTASI PCSGDKLEEK YVSWYQQRPG QSPVLVIYQD NKRPSGIPER FSGSNSGNTA TLTISGTQAM DEADYYCQVW DNYSVLFGGG TKLTVL(SEQ ID NO:79)

SYELTQPLSE SVAQGQTARI TCGGNNIESR NVHWYQQKPG QAPVLVIYRD NNRPSGIPER FSGSNSGNMA TLTISRAQAG DAADYYCQVW DNYTVLFGGG TKLTVL(SEQ ID NO:80)

SYELTQPPSV AVAPGKTARI TCGGNNIGSK NVHWYQQKPG QAPVLVIYRD SNRPSGIPER FSGSNSGNTA TLTISRAQAG DEADFYCQVW DNYIVLFGGG TKLTVL(SEQ ID NO:81)

QAVLTQPPSV SVAPGQTARI PCEGNNIGSK NVHWYRQKPG QVPVLVMYDD SDRPSGIPER FSGSNSGNTA TLTISGTQAM DEADYYCQVW DNYSVLFGGG TKLTVL(SEQ ID NO:82)

QPVLTQPLSV SVAPGQTARI TCGGNNIGSK NVHWYQQKPG QAPVLVIYRD SSRPSGIPER LSGSNSGDTA TLTISRAQAG DEADYYCQVW DDYIVVFGGG TKLTVL(SEQ ID NO:83)

SYELTQPPSV SVTPGQTATI TCGANDIGKR NVHWYQQRPG QSPVLVIYQD NKRPSGIPER FSGSNSGNTA TLTISGTQAM DEADYYCQVW DNYSVLFGGG TKLTVL(SEQ ID NO:84)

QPVLTQPSSV SVAPGQTATI SCGGHNIGSK NVHWYQQRPG QSPVLVIYQD NKRPSGIPER FSGSNSGNTA TLTISGTQAM DEADYYCQVW DNYSVLFGGG TKLTVL(SEQ ID NO:85)

in further embodiments, a humanized anti-CD 16 antibody, hCD16-M1 or a humanized anti-CD 16 antibody, the CD 16-binding domain of hCD16-M2 is complexed with a gp 41-binding domain to create a multispecific binding molecule capable of binding CD16 and gp 41. The amino acid sequence of the VH domain of hCD16-M1 (SEQ ID NO:127) is shown below (CDR NO:127)_HResidues are underlined):

EVQLVESGGG LVKPGGSLRL SCAASGFTFS NYGMSWVRQAPGKGLEWVAT ISGGGSYTFY PDSVKGRFTI SRDNAKNSLY LQMNSLRTEDTALYYCVRQS ARAPEPYWGQ GTLVTVSS

the amino acid sequence of the VL domain of hCD16-M1 (SEQ ID NO:128) is shown below (CDR)_LResidues are underlined):

DIQMTQSPSF LSASVGDRVT ITCRASQNVG THVAWYQQKP GKAPKSLLYSASYRYSGVPS RFSGSGSGTD FTLTISSLQS EDIATYYCQQ YKSYPLTFGQGTKLEIK

the antibody hCD16-M2 is a humanized derivative of the murine anti-human CD16 monoclonal antibody CD 16-M2. Humanization produced two suitable VH domains (hCD16-M2 VH 1and hCD16-M2VH 2), either of which could be used with the resulting humanized VL domain (hCD16-M2 VL 1).

The amino acid sequence (SEQ ID NO:129) of the VH domain of hCD16-M2VH 1 is shown below (CDR NO:129)_HResidues are underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSAMHWVRQAPGQGLEWMGY INHYNDGIKY NERFKGRVTI TADKSTSTAYMELSSLRSED TAVYYCATGY RYASWFASWG QGTLVTVSS

the amino acid sequence (SEQ ID NO:130) of the VH domain of hCD16-M2VH2 is shown below (CDR)_HResidues are underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT SSAMHWVRQAPGQGLEWMGY INHYNDGIKY NERFKGRVTI TADKSTSTAYMELSSLRSED TAVYYCA GY RYASWFASWG QGTLVTVSS

as will be appreciated, the amino acid sequence of hCD16-M2VH 1(SEQ ID NO:129) differs from the amino acid sequence of hCD16-M2VH 2(SEQ ID NO:130) in that it is in the CDRs_HThe residue before 3 had a T98R substitution (shown in box above).

The amino acid sequence of the VL domain of hCD16-M2 VL 1(SEQ ID NO:131) is shown below (CDR)_LResidues are underlined):

EIVLTQSPAT LSVSPGERAT LSCRASQNIG TSIHWYQQKP DQSPKLLIKSVSESISGVPS RFSGSGSGTD FTLTINSLEA EDFATYYCQQ SNSWPLTFGQGTKLEIK

additional anti-CD 16 antibodies that may be used include, but are not limited to, The commercially available antibodies DJ130c (Tamm, A. et al (1996) "The Binding Epitopes Of Human CD16(Fc Gamma RIII) Monoclonal antibodies.Immunodizations For Ligand Binding," J.Immunol.157(4):1576 1581), eBioCB16(ThermoFisher) or 1D3(Abcam), or those described in PCT publications WO 03/101485 and WO 2006/125668.

E. Exemplary TCR binding Capacity

In one embodiment, the molecule of the invention is capable of binding an epitope of a T Cell Receptor (TCR). The T cell receptor is naturally expressed by CD4+ T cells or CD8+ T cells and allows such cells to recognize antigenic peptides bound and presented by MHC class I or class II proteins of antigen presenting cells. TCR Recognition Of the pMHC (Peptide-MHC) complex initiates the spread Of a cellular Immune response that results In cytokine production And lysis Of antigen presenting cells (see, e.g., Armstrong, K.M. et al (2008) "structural Changes And Flexibility In T-Cell Receptor Recognition Of Peptide-MHC Complexes," biochem. J.415(Pt 2): 183. 196; Willemsen, R. (2008) "Selection Of Human Antibody Fragments" Molecular T-Cell expression For additive T-Cell Therapy, "Cytometry A.73(11): 1093. 1099; Beier, K.C. et al (Master T-switch Activation analysis. 12)," Receptor T-Cell Activation J.11. 12. expression Of protein J.11., "Receptor T-Cell Receptor Therapy"; protein, K.C. et al "(Master T-switch T-Cell Activation) Activation expression J.12; expression Of protein J.12. 12. expression Of protein kinase J.11.), "Immunology 129(2): 170-177; guy, C.S. et al (2009) "Organization Of Proximal Signal Initiation At The TCR: CD3 Complex," immunological. Rev.232(1): 7-21; St.Clair, E.W. (Epub 2009Oct 12) "Novel Targeted therapeutics For Autoimmitude," Current. Opin. Immunol.21(6): 648-657; baeuuerle, P.A. et al (Epub 2009Jun 9) "Bispecific T-Cell Engaging Antibodies For Cancer Therapy," Cancer Res.69(12): 4941-; Smith-Garvin, J.E. et al (2009) "T Cell Activation," Annu.Rev.Immunol.27: 591-one 619; renderers, L. et al (2003) "Engineered CD3 Antibodies For immunological delivery," Clin. Exp. Immunol.133(3): 307-.

Molecules that specifically bind to T cell receptors include the anti-TCR antibody "BMA 031". (EP 0403156; Kurrle, R. et al (1989) "BMA 031-A TCR-Specific Monoclonal Antibody For Clinical Application," Transplant Proc.21(1Pt 1): 1017-.

The amino acid sequence of the VH domain of BMA 031 (SEQ ID NO:86) is shown below (CDR)_HResidues are underlined):

QVQLVQSGAE VKKPGASVKV SCKASGYKFT SYVMHWVRQA PGQGLEWIGY INPYNDVTKY NEKFKGRVTI TADKSTSTAY LQMNSLRSED TAVHYCARGS YYDYDGFVYW GQGTLVTVSS

the amino acid sequence of the VL domain of BMA 031 (SEQ ID NO:87) is shown below (CDR)_LResidues are underlined):

EIVLTQSPAT LSLSPGERAT LSCSATSSVS YMHWYQQKPG KAPKRWIYDT SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SSNPLTFGQG TKLEIK

F. exemplary NKG2D binding Capacity

In one embodiment, the molecule of the invention is capable of binding to an epitope of the NKG2D receptor. The NKG2D Receptor is expressed on all human (And other mammalian) Natural killer Cells (Bauer, S. et al (1999) "Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-induced MICA," Science 285(5428): 727-729; Jamieson, A.M. et al (2002) "The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing," Immunity17(1):19-29) And on all CD8⁺Expressed On T Cells (Groh, V.et al (2001) "stimulation Of CD 8. alpha. beta. T Cells By NKG2D Via Engament By MIC Induced On Virus-Infected Cells," nat. Immunol.2(3): 255-260; Jamieson, A.M. et al (2002) "The Role Of The NKG2D Immunorecter In Immunity Cell Activation And Natural packaging," Immunity17(1): 19-29). Molecules that specifically bind the NKG2D receptor include the anti-NKG 2D antibodies "KYK-1.0" and "KYK-2.0". (Kwong, KY et al (2008) "Generation, Affinity mapping, And analysis Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antibody And Activity," J.mol.Biol.384:1143-1156)。

The amino acid sequence (SEQ ID NO:88) of the VH domain of KYK-1.0 is shown below (CDR)_HResidues are underlined):

EVQLVESGGG VVQPGGSLRL SCAASGFTFS SYGMHWVRQAPGKGLEWVAF IRYDGSNKYY ADSVKGRFTI SRDNSKNTKYLQMNSLRAED TAVYYCAKDR FGYYLDYWGQ GTLVTVSS

the amino acid sequence (SEQ ID NO:89) of the VL domain of KYK-1.0 is shown below (CDR)_LResidues are underlined):

QPVLTQPSSV SVAPGETARI PCGGDDIETK SVHWYQQKPG QAPVLVIYDDDDRPSGIPER FFGSNSGNTA TLSISRVEAG DEADYYCQVWDDNNDEWVFG GGTQLTVL

the amino acid sequence (SEQ ID NO:90) of the VH domain of KYK-2.0 is shown below (CDR)_HResidues are underlined):

QVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMHWVRQAPGKGLEWVAF IRYDGSNKYY ADSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCAKDR GLGDGTYFDY WGQGTTVTVS S

the amino acid sequence (SEQ ID NO:91) of the VL domain of KYK-2.0 is shown below (CDR)_LResidues are underlined):

QSALTQPASV SGSPGQSITI SCSGSSSNIG NNAVNWYQQL PGKAPKLLIYYDDLLPSGVS DRFSGSKSGT SAFLAISGLQ SEDEADYYCA AWDDSLNGPVFGGGTKLTVL

G. exemplary NKp46 binding Capacity

In one embodiment, the molecule of the invention is capable of binding to an epitope of NKp46(CD 335). NKp46 is the major NK Cell-activating receptor involved in the elimination of target cells (Sivori S, et al (1997) 'p 46, aNovel Natural Killer Cell-specific Surface Mobile at media Cell Activation,' J.Exp.Med.186 (7): 1129-36.). NKp46 is uniquely expressed on all NK cell subsets (Narni-Mancinelli E, et al (2011), "site mapping analysis of lymphoid cells expressing the NKp46 cell surface receiver," Proc Natl Acad Sci U S A108: 18324-9). Molecules that specifically bind NKp46 include the anti-NKp 46 antibodies "BAB 281" and "NKp 46-3" (WO 2015/197593).

The amino acid sequence of the VH domain of BAB281 (SEQ ID NO:92) is shown below (CDR)_HResidues are underlined):

QIQLVQSGPE LQKPGETVKI SCKASGYTFT NYGMNWVKQA PGKGLKWMGW INTNTGEPTY AEEFKGRFAF SLETSASTAY LQINNLKNED TATYFCARDY LYYFDYWGQG TTLTVSS

the amino acid sequence of the VL domain of BAB281 (SEQ ID NO:93) is shown below (CDR)_LResidues are underlined):

DNIVMTQSPK SMSMSVGERV TLTCKASENV VTYVSWYQQK PEQSPKLLIY GASNRYTGVP DRFTGSGSAT DFTLTISSVQ AEDLADYHCG QGYSYPYTFG GGTKLEIK

the amino acid sequence of the VH domain of NKp46-3 (SEQ ID NO:94) is shown below (CDR)_HResidues are underlined):

EVQLQQSGPE LVKPGASVKI SCKTSGYTFT EYTMHWVKQS HGKSLEWIGG ISPNIGGTSY NQKFKGKATL TVDKSSSTAY MELRSLTSED SAVYYCARRG GSFDYWGQGT TLTVSS

the amino acid sequence of the VL domain of NKp46-3 (SEQ ID NO:95) is shown below (CDR)_LResidues are underlined):

DIVMTQSPAT LSVTPGDRVS LSCRASQSIS DYLHWYQQKS HESPRLLIKY ASQSISGIPS RFSGSGSGSD FTLSINSVEP EDVGVYYCQN GHSFPLTFGA GTKLELK

additional antibodies that bind NKp46 are described in WO2015/197593 and WO 2017/016805. Other exemplary antibodies that bind to the cell surface of natural killer cells include antibodies: a1, AC2, EPR3678(2), EPR20461, EPR20627 and IMG17B5F11 (which binds CD 39); TB01, HNK-1/Leu-7 and NK1 (which binds CD 57); FN50 (which binds CD 69); 5B5, B-L2, TS82B and C33 (which binds CD 82); 3B3, B199.2 and EP7169 (which binds CD 161); 17D9 (which binds CLEC 1B); 2F9 (which binds KIR2DL 1); EPR8825 (which binds KIR2DL 2); mAb 33 (which binds KIR2DL 4); 11E3, 17B4, EPR4392(2), EPR20261 and EPR20627 (which binds to lymphocyte activator gene 3); a10, C7, CX5, 1D11 and MM0489-10R27 (which binds NKG 2D); BMK13 (which binds PRG 2); EPR9916 (which binds SLAMF6), and the like. Antibodies capable of binding to each such NK cell surface molecule are commercially available from Abcam plc and other sources, and can be readily adapted for the purposes of the present invention.

Exemplary alternative HIV-1 binding molecules

The mature human immunodeficiency virus type 1(HIV-1) envelope (Env) glycoprotein trimer comprises three copies of the non-covalently linked gp120/gp41 heterodimer resulting from cleavage of the viral gp160 precursor protein. In one embodiment, the molecule of the invention is capable of binding to an epitope of gp120, gp160 and/or gp41 of the HIV-1Env protein which is different from the epitope of 7B 2.

Monoclonal Antibody A32 Recognizes a Conformational Epitope In The C1 region Of HIV-1Env gp120 (Wyatt et al (1995) "inversion Of The V1/V2 Variable Loop Structure In The expression Of Human Immunodeficiency Virus Type 1gp120 Epitopes Induced By Receptor Binding," J.Virol.69: 5723. 5733) and Mediates Potent ADCC Activity as well as detectable ADCC-mediated Ab Activity In individuals That block a comparable proportion Of HIV-1 infection (Ferrari, G. et al (2011) "Ann HIV-1gp120 Envelope monomer Antibody sites antibodies C1 structural antibodies mediated ADCC-mediated ADCC 7085. J.14. ADCC-mediated ADCC-Activity).

A variety of VH domains of antibody A32 have been reported in the art with minimal changes in the reported framework regions 1 and/or 4 (see, e.g., protein database accession numbers PDB: 4YBL _ H, US2015/0239961 and WO 2006/044410). Any of these variant antibody A32VH domains may be used in accordance with the present invention. The amino acid sequence (SEQ ID NO:96) of an illustrative VH domain of A32 is shown below (CDR)_HResidues are underlined):

QVQLQESGPG LVKPSQTLSL SCTVSGGSSS SGAHYWSWIRQYPGKGLEWI GYIHYSGNTY YNPSLKSRIT ISQHTSENQF SLKLNSVTVA DTAVYYCARG TRLRTLRNAF DIWGQGTLVT VSS

the amino acid sequence of the VL domain of A32 (SEQ ID NO:97) is shown below (CDR)_LUnderlining of residuesLine shows):

QSALTQPPSA SGSPGQSVTI SCTGTSSDVG GYNYVSWYQH HPGKAPKLII SEVNNRPSGV PDRFSGSKSG NTASLTVSGL QAEDEAEYYC SSYTDIHNFV FGGGTKLTVL

the amino acid sequence of the VL domain of A32 (SEQ ID NO:97) can be used with the illustrative VH domain of A32 (SEQ ID NO:96) or with any variant antibody A32VH domain (see, e.g., protein database accession numbers PDB: 4YBL _ H, US2015/0239961 and WO 2006/044410) to form an anti-HIV-1 Env gp120 epitope binding site.

Monoclonal Antibody 10-1074 targets the basal portion of the V3 loop of HIV-1Env gp120 (see, e.g., WO 2014/063059) and is among the most potent isolated anti-HIV-1 neutralizing antibodies, and has shown some In vivo activity In early clinical trials (Caskey, M., et al, (2017) "antibodies 10-1074 supresses Viremia In HIV-1-infected individuals," Nat Med.23: 185-191).

The amino acid sequence of the VH domain of 10-1074 (SEQ ID NO:98) is shown below (CDR)_HResidues are underlined):

QVQLQESGPG LVKPSETLSV TCSVSGDSMN NYYWTWIRQS PGKGLEWIGY ISDRESATYN PSLNSRVVIS RDTSKNQLSL KLNSVTPADT AVYYCATARR GQRIYGVVSF GEFFYYYSMD VWGKGTTVTV SS

the amino acid sequence of the VL domain of 10-1074 (SEQ ID NO:99) is shown below (CDR)_LResidues are underlined):

SYVRPLSVAL GETARISCGRQALGSRAVQW YQHRPGQAPI LLIYNNQDRP SGIPERFSGT PDINFGTRAT LTISGVEAGD EADYYCHMWD SRSGFSWSFG GATRLTVL

monoclonal Antibody 3BNC117 targets the CD4 binding site of gp120 and is a Broadly Neutralizing anti-HIV-1 Antibody (see, e.g., WO 2013/016468) that has shown some In vivo activity In early clinical trials (Caskey, M., et al (2015) "Virus Suppressed In HIV-1-Infected Humans By Broadly neutral Antibody 3BNC117," Nature 522: 487-.

The amino acid sequence of the VH domain of 3BNC117 (SEQ ID NO:100) is shown below (CDR)_HResidues are underlined):

QVQLLQSGAA VTKPGASVRV SCEASGYNIR DYFIHWWRQA PGQGLQWVGW INPKTGQPNN PRQFQGRVSL TRHASWDFDT FSFYMDLKAL RSDDTAVYFC ARQRSDYWDFDVWGSGTQVT VSS

the amino acid sequence of the VL domain of 3BNC117 (SEQ ID NO:101) is shown below (CDR)_LResidues are underlined):

DIQMTQSPSS LSASVGDTVT ITCQANGYLN WYQQRRGKAP KLLIYDGSKL ERGVPSRFSG RRWGQEYNLT INNLQPEDIA TYFCQVYEFV VPGTRLDLK

monoclonal Antibodies PGT121 And PGT145 are broadly Neutralizing HIV-1 Antibodies that depend primarily on gp120 glycans for Env recognition (Mouquet H, et al, (2012) "Complex-type N-glycan recognition by cell branched HIV Antibodies", Proc Natl Acad Sci U S A109: E3268-3277; Yasmen, A., et al (2014) "Differential Binding Of neutral Binding Non-neutral Antibodies A-neutral HIV-1Env Trimers, Uncleavad Env Proteins, And d 2012 monoclonal Subunirology" Retrovirology 11: 41; WO/030904).

The amino acid sequence (SEQ ID NO:102) of the VH domain of PGT121 is shown below (CDR)_HResidues are underlined):

QMQLQESGPG LVKPSETLSL TCSVSGASIS DSYWSWIRRS PGKGLEWIGY VHKSGDTNYS PSLKSRVNLS LDTSKNQVSL SLVAATAADS GKYYCARTLH GRRIYGIVAF NEWFTYFYMD VWGNGTQVTVSS

the amino acid sequence of the VL domain of PGT121 (SEQ ID NO:103) is shown below (CDR)_LResidues are underlined):

SDISVAPGET ARISCGEKSL GSRAVQWYQH RAGQAPSLII YNNQDRPSGI PERFSGSPDS PFGTTATLTI TSVEAGDEAD YYCHIWDSRV PTKWVFGGGT TLTVL

the amino acid sequence of the VH domain of PGT145 (SEQ ID NO:104) is shown below (CDR)_HResidues are underlined):

QVQLVQSGAE VKKPGSSVKV SCKASGNSFS NHDVHWVRQA TGQGLEWMGW MSHEGDKTGL AQKFQGRVTI TRDSGASTVY MELRGLTADD TAIYYCLTGS KHRLRDYFLY NEYGPNYEEW GDYLATLDVW GHGTAVTVSS

the amino acid sequence of the VL domain of PGT145 (SEQ ID NO:105) is shown below (CDR)_LResidues are underlined):

EVVITQSPLF LPVTPGEAAS LSCKCSHSLQ HSTGANYLAW YLQRPGQTPR LLIHLATHRASGVPDRFSGS GSGTDFTLKI SRVESDDVGT YYCMQGLHSP WTFGQGTKVE IK

the Monoclonal Antibody VRC01 is a Broadly Neutralizing anti-HIV-1 Antibody directed against the CD4 binding site Of gp120 (Wu, X. et al (2010) "random Design Of Envelope identities Broad neutral Human Monoclonal Antibodies To HIV-1," Science 329:856-861And Zhou, T. et al (2010) "Structural base For Broad content catalysis Of HIV-1By Antibody VRC01," Science 329: 811-817; WO 2013/163427).

The amino acid sequence of the VH domain of VRC01 (SEQ ID NO:106) is shown below (CDR)_HResidues are underlined):

QVQLVQSGGQ MKKPGESMRI SCRASGYEFI DCTLNWIRLA PGKRPEWMGW LKPRGGAVNY ARPLQGRVTM TRDVYSDTAF LELRSLTVDD TAVYFCTRGK NCDYNWDFEH WGRGTPVIVS S

the amino acid sequence of the VL domain of VRC01 (SEQ ID NO:107) is shown below (CDR)_LResidues are underlined):

EIVLTQSPGT LSLSPGETAI ISCRTSQYGS LAWYQQRPGQ APRLVIYSGS TRAAGIPDRF SGSRWGPDYN LTISNLESGD FGVYYCQQYE FFGQGTKVQV DIKR

monoclonal antibody 10E8 is a broadly neutralizing HIV-1gp41 membrane-adjacent outer region (MPER) -Specific antibody (Huang, J., et al 2012, "Broad And Point mutagenesis Of HIV-1By A gp41-Specific Human antibody," Nature.491: 406-12).

The amino acid sequence of the VH domain of 10E8 (SEQ ID NO:108) is shown below (CDR)_HResidues are underlined):

EVQLVESGGG LVKPGGSLRL SCSASGFDFD NAWMTWVRQP PGKGLEWVGR ITGPGEGWSV DYAAPVEGRF TISRLNSINF LYLEMNNLRM EDSGLYFCAR TGKYYDFWSG YPPGEEYFQD WGRGTLVTVS S

the amino acid sequence of the VL domain of 10E8 (SEQ ID NO:109) is shown below (CDR)_LResidues are underlined):

SYELTQETGV SVALGRTVTI TCRGDSLRSH YASWYQKKPG QAPILLFYGK NNRPSGVPDR FSGSASGNRA SLTISGAQAE DDAEYYCSSR DKSGSRLSVF GGGTKLTVL

the invention specifically includes and encompasses VL domains and/or VH domains comprising the anti-HIV-1 monoclonal antibodies provided above, and/or 1, 2 or all 3 CDRs of the VL region_LAnd/or 1, 2 or all 3 CDRs of a VH domain_HIs a molecule that binds gp41 multispecific.

Exemplary multispecific gp 41-binding molecules

Provided herein are a series of exemplary gp 41-binding molecules that incorporate a first epitope-binding domain that is immunospecific for an epitope of gp41 (i.e., binds to the domain of gp 41) and a second epitope-binding domain that is immunospecific for an epitope of ECM. Optionally such molecules incorporate a third epitope-binding domain (or a third epitope-binding domain and a fourth epitope-binding domain) that is immunospecific for a different epitope of gp41 and/or an epitope of a different HIV-1 molecule and/or an epitope of an ECM and/or an epitope of a different ECM. Particularly preferred are such molecules comprising an optimized gp 41-binding domain of 7B2 GL. The structure and sequence of this illustrative gp 41-binding molecule is summarized in table 7 and described in detail below. As will be appreciated, similar molecules may be similarly constructed (by employing the VL and VH domains of the desired antibody in place of the VL and VH domains used in the illustrative gp 41-binding molecule).

Gp41 x CD3 binding molecule, DART-1

Gp41 x CD 3-binding molecules designated "DART-1" were the first illustrative bispecific gp 41-binding molecules. DART-1 is a bispecific diabody containing Fc domains capable of binding to gp41 and CD3 antigens. DART-1 consists of three polypeptide chains and possesses one binding domain comprising the VL and VH domains of anti-human gp41 antibody 7B2 (and thus immunospecific for an epitope of gp 41) and one binding domain comprising the VL and VH domains of CD3mAb1 (and thus immunospecific for an epitope of CD3 antigen). The three polypeptide chains associate to form a covalently bound diabody that is capable of immunospecifically binding to an epitope of gp41 and an epitope of CD3 antigen (see, e.g., fig. 4A).

The first polypeptide chain of DART-1 has the amino acid sequence of SEQ ID NO: 110: DIVMTQSPDS LAVSPGERAT IHCKSSQTLL YSSNNRHSIA WYQQRPGQPPKLLLYWASMR LSGVPDRFSG SGSGTDFTLT INNLQAEDVA IYYCHQYSSHPPTFGHGTRV EIKGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTFSTYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGRFTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYVSWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPEVTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Residues 1-113 of the first polypeptide chain of DART-1 (SEQ ID NO:110) correspond to the VL domain of 7B 2(SEQ ID NO: 55). Residue 114-121 (double underlined) of the first polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain correspond to the VH domain (SEQ ID NO:63) of CD3mAb1 (D65G). Residue 247-251 corresponds to the linker (SEQ ID NO:21, underlined). Residue 252-279 of the first polypeptide chain corresponds to the cysteine-containing E-helix (SEQ ID NO: 31). Residues 280-292 of the first polypeptide chain correspond to the linker (SEQ ID NO: 40). Residues 293-509 of the first polypeptide chain correspond to the "carrying pestle" (SEQ ID NO:48) in which the last residue is lysine.

The second polypeptide chain of DART-1 has the amino acid sequence of SEQ ID NO: 111: QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLIGGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYCALWYSNLWVF GGGTKLTVLGGGGSGGGGQV QLVQSGGGVFKPGGSLRLSC EASGFTFTEY YMTWVRQAPG KGLEWLAYIS KNGEYSKYSPSSNGRFTISR DNAKNSVFLQ LDRLSADDTA VYYCARADGL TYFSELLQYIFDLWGQGARV TVSSASTKGK VAACKEKVAA LKEKVAALKE KVAALKE

Residues 1-110 of the second polypeptide chain of DART-1 (SEQ ID NO:111) correspond to the VL domain of CD3mAb 1(SEQ ID NO: 62). Residue 111-118 (double underlined) of the second polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-244 of the second polypeptide chain correspond to the VH domain of 7B 2(SEQ ID NO: 56). Residues 245-249 of the second polypeptide chain correspond to the linker (SEQ ID NO:21, underlined). Residues 250-277 of the second polypeptide chain correspond to the cysteine-containing K-helix (SEQ ID NO: 32).

The third polypeptide chain of DART-1 has the amino acid sequence of SEQ ID NO: 112: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLHQDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTKNQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKLTVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK

Residues 1-10 of the third polypeptide chain of DART-1 (SEQ ID NO:112) correspond to linker (SEQ ID NO: 40). Residues 11-227 of the third polypeptide chain correspond to the IgG1 CH2-CH3 domain (SEQ ID NO:50) carrying the mortar containing the H435R substitution (shown underlined) and wherein the last residue is lysine. As described above, the H435R substitution abolished the ability of the molecule to bind to binding protein a.

As will be appreciated, the third polypeptide chain of DART-1 does not contain any epitope binding site and therefore can be employed in a variety of gp 41-binding molecules having the overall structure provided in fig. 4A-4B. Accordingly, the third polypeptide chain of DART-1 is referred to herein as a "normal double antibody polypeptide chain".

Gp41 x CD3 binding molecule, DART-A

A second illustrative gp41 x CD3 binding molecule designated "DART-a" is similar to DART-1 described above, but contains the VL and VH domains of anti-human gp41 antibody 7B2GL in place of the parent 7B2VL and VH domains.

The first polypeptide chain of DART-A has the amino acid sequence of SEQ ID NO 113: DIVMTQSPDS LAVSLGERAT INCKSSQTLL YSSNNRHSIA WYQQKPGQPP KLLIYWASMR LSGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCHQYSSH PPTFGQGTKV EIKGGGSGGG GEVQLVESGG GLVQPGGSLR LSCAASGFTF STYAMNWVRQ APGKGLEWVG RIRSKYNNYA TYYADSVKGR FTISRDDSKN SLYLQMNSLK TEDTAVYYCV RHGNFGNSYV SWFAYWGQGT LVTVSSASTK GEVAACEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPEVTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

Residues 1-113 of the first polypeptide chain of DART-A (SEQ ID NO:113) correspond to the VL domain of 7B2GL (SEQ ID NO: 57). Residue 114-121 (double underlined) of the first polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain correspond to the VH domain (SEQ ID NO:63) of CD3mAb1 (D65G). Residue 247-251 corresponds to the linker (SEQ ID NO:21, underlined). Residue 252-279 of the first polypeptide chain corresponds to the cysteine-containing E-helix (SEQ ID NO: 31). Residues 280-292 of the first polypeptide chain correspond to the linker (SEQ ID NO: 40). Residues 293-509 of the first polypeptide chain correspond to "knob-carrying" (SEQ ID NO:48), with the last residue being lysine.

The second polypeptide chain of DART-A has the amino acid sequence of SEQ ID NO: 114: QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLIGGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYCALWYSNLWVF GGGTKLTVLGGGGSGGGGQV QLVESGGGLVKPGGSLRLSC AASGFTFSEY YMTWIRQAPG KGLEWVSYIS KNGEYSKYSPSSNGRFTISR DNAKNSLYLQ MNSLRAEDTA VYYCARADGL TYFSELLQYIFDLWGQGTLV TVSSASTKGK VAACKEKVAA LKEKVAALKE KVAALKE

Residues 1-110 of the second polypeptide chain of DART-A (SEQ ID NO:114) correspond to the VL domain of CD3mAb 1(SEQ ID NO: 62). Residue 111-118 (double underlined) of the second polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residue 119-244 of the second polypeptide chain corresponds to the VH domain of 7B2GL (SEQ ID NO: 58). Residues 245-249 of the second polypeptide chain correspond to the linker (SEQ ID NO:21, underlined). Residues 250-277 of the second polypeptide chain correspond to the cysteine-containing K-helix (SEQ ID NO: 32).

The amino acid sequence of the third polypeptide chain of DART-A is identical to that of DART-1 (i.e., SEQ ID NO: 112).

Gp41 x CD16 binding molecule, DART-B

Gp41 x CD 16-binding molecules designated "DART-B" are third illustrative bispecific gp 41-binding molecules. DART-B is similar to DART-A described above, but includes a domain that binds CD16 in place of the domain that binds CD3 of DART-A. Thus, DART-B consists of three polypeptide chains and possesses one binding domain comprising the VL domain and VH domain of anti-human gp41 antibody 7B2GL (and thus immunospecific for an epitope of gp 41), and one binding domain comprising the VL domain and VH domain of anti-human CD16 antibody 3G8 (and thus immunospecific for an epitope of CD 16).

The first polypeptide chain of DART-B has the amino acid sequence of SEQ ID NO: 115: DIVMTQSPDS LAVSLGERAT INCKSSQTLL YSSNNRHSIA WYQQKPGQPP KLLIYWASMR LSGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCHQYSSH PPTFGQGTKV EIKGGGSGGG GQVTLRESGP ALVKPTQTLT LTCTFSGFSL STSGMGVGWI RQPPGKALEW LAHIWWDDDK RYNPALKSRL TISKDTSKNQ VVLTMTNMDP VDTATYYCAQ INPAWFAYWG QGTLVTVSSA STKGEVAACE KEVAALEKEV AALEKEVAAL EKGGGDKTHT CPPCPAPEAA GGPSVFLFPP KPKDTLMISR TPEVTCVVVDVSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSREEMTKNQVSL WCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

Residues 1-113 of the first polypeptide chain of DART-B (SEQ ID NO:115) correspond to the VL domain of 7B2GL (SEQ ID NO: 57). Residue 114-121 (double underlined) of the first polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-239 of the first polypeptide chain correspond to the VH domain of h3G8 (SEQ ID NO: 74). Residue 240-244 corresponds to the linker (SEQ ID NO:21, underlined). Residues 245-272 of the first polypeptide chain correspond to the cysteine-containing E-helix (SEQ ID NO: 31). Residue 273-285 of the first polypeptide chain corresponds to the linker (SEQ ID NO: 40). Residue 286-502 of the first polypeptide chain corresponds to the "carrying knob" (SEQ ID NO:48) where the last residue is lysine.

The second polypeptide chain of DART-B has the amino acid sequence of SEQ ID NO: 116: DIVMTQSPDS LAVSLGERAT INCKASQSVD FDGDSFMNWY QQKPGQPPKLLIYTTSNLES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQSNEDPYTFGQGTKLEI KGGGSGGGGQ VQLVESGGGL VKPGGSLRLS CAASGFTFSEYYMTWIRQAP GKGLEWVSYI SKNGEYSKYS PSSNGRFTIS RDNAKNSLYLQMNSLRAEDT AVYYCARADG LTYFSELLQY IFDLWGQGTL VTVSSASTKGKVAACKEKVA ALKEKVAALK EKVAALKE

Residues 1-111 of the second polypeptide chain of DART-B (SEQ ID NO:116) correspond to the VL domain of h3G8 (SEQ ID NO: 75). Residue 112-119 (double underlined) of the second polypeptide chain corresponds to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residue 120-245 of the second polypeptide chain corresponds to the VH domain of 7B2GL (SEQ ID NO: 58). Residue 246-250 of the second polypeptide chain corresponds to the linker (SEQ ID NO:21, underlined). Residue 251-278 of the second polypeptide chain corresponds to the cysteine-containing K-helix (SEQ ID NO: 32).

The amino acid sequence of the third polypeptide chain of DART-B is identical to that of DART-1 (i.e., SEQ ID NO: 112).

Gp41 xCD 3xCD 8 binding molecule, TRIDENT-A

Gp41 x CD3x CD8 binding molecules designated "TRIDENT-A" are the first illustrative trivalent binding molecules to gp 41. TRIDENT-a consists of four polypeptide chains and possesses one binding domain comprising the VL and VH domains of anti-human gp41 antibody 7B2GL (and thus immunospecific for an epitope of gp 41), one binding domain comprising the VL and VH domains of CD3mAb1 (and thus immunospecific for an epitope of CD3 antigen), and one binding domain comprising the VL and VH domains of TRX8 (and thus immunospecific for an epitope of CD8 antigen). The four polypeptide chains associate to form a covalently bound trivalent molecule capable of immunospecifically binding to an epitope of gp41, an epitope of CD3 antigen, and an epitope of CD8 (see, e.g., fig. 6A).

The amino acid sequence of the first polypeptide chain of TRIDENT-A is identical to the amino acid sequence of the first polypeptide chain of DART-A diabody described above (SEQ ID NO: 113). Similarly, the amino acid sequence of the second polypeptide chain of TRIDENT-A is identical to the amino acid sequence of the second polypeptide chain of DART-A diabody described above (SEQ ID NO: 114).

The third polypeptide chain of TRIDENT-A has the amino acid sequence of SEQ ID NO: 117: QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMNWVRQAPGKGLEWVAL IYYDGSNKFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCAKPH YDGYYHFFDS WGQGTLVTVS SASTKGPSVF PLAPSSKSTSGGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVVTVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEAAGGPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHNAKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTISKAKGQPREP QVYTLPPSRE EMTKNQVSLS CAVKGFYPSD IAVEWESNGQPENNYKTTPP VLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VMHEALHNRYTQKSLSLSPG K

Residues 1-121 of the third polypeptide chain of TRIDENT-A correspond to the VH domain of anti-CD 8 antibody TRX 2(SEQ ID NO: 70). Residues 121-219 correspond to the IgG1 CH1 domain (SEQ ID NO: 3). Residues 220-234 correspond to the IgG1 hinge domain (SEQ ID NO: 7). Residue 235-451 corresponds to the CH2-CH3 domain of IgG1 "carrying the mortar" (SEQ ID NO: 50).

The fourth polypeptide chain of TRIDENT-A has the amino acid sequence of SEQ ID NO: 118: DIQMTQSPSS LSASVGDRVT ITCKGSQDIN NYLAWYQQKP GKAPKLLIYNTDILHTGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCYQ YNNGYTFGQGTKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVDNALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGLSSPVTKSFNR GEC

Residues 1-106 of the fourth polypeptide chain of TRIDENT-A correspond to the VL domain of anti-CD 8 antibody TRX 2(SEQ ID NO: 71). Residue 107-213 corresponds to the CL kappa domain (SEQ ID NO: 1).

Gp41 x CD3x gp120 binding molecule, TRIDENT-B

A second illustrative trivalent gp41 x CD3x gp120 binding molecule designated "TRIDENT-B" is similar to TRIDENT-A described above, but contains the VL domain and the VH domain of anti-human gp120 antibody A32 in place of the TRX8 VL domain and the VH domain of TRIDENT-A. Thus, TRIDENT-B consists of four polypeptide chains and possesses one binding domain comprising the VL domain and the VH domain of anti-human gp41 antibody 7B2GL (and thus immunospecific for an epitope of gp 41), one binding domain comprising the VL domain and the VH domain of CD3mAb1 (and thus immunospecific for an epitope of CD3 antibody) and one binding domain comprising the VL domain and the VH domain of a32 (and thus immunospecific for an epitope of gp120 antibody).

The amino acid sequence of the first polypeptide chain of TRIDENT-B is identical to the amino acid sequence of the first polypeptide chain of DART-A diabody described above (SEQ ID NO: 113). Similarly, the amino acid sequence of the second polypeptide chain of TRIDENT-B is identical to the amino acid sequence of the second polypeptide chain of DART-A diabody described above (SEQ ID NO: 114).

Amino acid sequence of SEQ ID NO 119 of the third polypeptide chain of TRIDENT-B: QVQLQESGPG LVKPSQTLSL SCTVSGGSSS SGAHYWSWIR QYPGKGLEWIGYIHYSGNTY YNPSLKSRIT ISQHTSENQF SLKLNSVTVA DTAVYYCARGTRLRTLRNAF DIWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGCLVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLGTQTYICNVNH KPSNTKVDKR VEPKSCDKTH TCPPCPAPEA AGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREEQYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPREPQVYTLPPS REEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN RYTQKSLSLSPGK

Residues 1-123 of the third polypeptide chain of TRIDENT-B correspond to the VH domain of anti-gp 120 antibody A32 (SEQ ID NO: 96). Residue 124-221 corresponds to the IgG1 CH1 domain (SEQ ID NO: 3). Residues 222-236 corresponded to the IgG1 hinge domain (SEQ ID NO: 7). Residues 237 and 453 correspond to the CH2-CH3 domain of IgG1 "carrying the mortar" (SEQ ID NO: 50).

The fourth polypeptide chain of TRIDENT-B has the amino acid sequence of SEQ ID NO: 120: QSALTQPPSA SGSPGQSVTI SCTGTSSDVG GYNYVSWYQH HPGKAPKLIISEVNNRPSGV PDRFSGSKSG NTASLTVSGL QAEDEAEYYC SSYTDIHNFVFGGGTKLTVL RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC

Residues 1-110 of the fourth polypeptide chain of TRIDENT-B correspond to the VL domain of anti-gp 120 antibody A32 (SEQ ID NO: 97). Residue 111-217 corresponds to the CL kappa domain (SEQ ID NO: 1).

Gp41 x CD16 x gp120 binding molecule, TRIDENT-C

A third illustrative trivalent gp41 x CD16 x gp120 binding molecule designated "TRIDENT-C" is similar to the TRIDENT-B described above, but contains the VL and VH domains of anti-human CD16 antibody h3G8 in place of the CD3mAb 1(D65G) VL and VH domains of TRIDENT-B. Thus, TRIDENT-C consists of four polypeptide chains and is provided with one binding domain comprising the VL domain and VH domain of anti-human gp41 antibody 7B2GL (and thus immunospecific for an epitope of gp 41), one binding domain comprising the VL domain and VH domain of h3G8 (and thus immunospecific for an epitope of the CD16 antigen) and one binding domain comprising the VL domain and VH domain of a32 (and thus immunospecific for an epitope of gp 120).

The amino acid sequence of the first polypeptide chain of TRIDENT-C is identical to the amino acid sequence of the first polypeptide chain of DART-B diabody described above (SEQ ID NO: 115). Similarly, the amino acid sequence of the second polypeptide chain of TRIDENT-C is identical to the amino acid sequence of the second polypeptide chain of DART-B diabody described above (SEQ ID NO: 116).

The amino acid sequences of the third and fourth polypeptides of TRIDENT-C are identical to the amino acid sequences of the third and fourth polypeptides of TRIDENT-B described above (SEQ ID NO:119 and SEQ ID NO: 120).

Gp41 x CD3x gp41 binding molecule, TRIDENT-D

A third illustrative trivalent gp41 x CD3x gp41 binding molecule designated "TRIDENT-D" is similar to the TRIDENT-A described above, but contains the VL and VH domains of anti-human gp41 antibody 7B2GL in place of the TRX2 anti-CD 8 VL and VH domains of TRIDENT-A. Thus, TRIDENT-D consists of four polypeptide chains and is provided with two binding domains including the VL domain and the VH domain of anti-human gp41 antibody 7B2GL (and thus immunospecific for an epitope of gp 41), one binding domain including the VL domain and the VH domain of hCD 3mAb1 (and thus immunospecific for an epitope of CD3 antibodies).

The amino acid sequence of the first polypeptide chain of TRIDENT-D is identical to the amino acid sequence of the first polypeptide chain of DART-A diabody described above (SEQ ID NO: 113). Similarly, the amino acid sequence of the second polypeptide chain of TRIDENT-D is identical to the amino acid sequence of the second polypeptide chain of DART-A diabody described above (SEQ ID NO: 114).

The third polypeptide chain of TRIDENT-D has the amino acid sequence of SEQ ID NO: 121: QVQLVESGGG LVKPGGSLRL SCAASGFTFS EYYMTWIRQA PGKGLEWVSYISKNGEYSKY SPSSNGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARADGLTYFSELLQ YIFDLWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAALGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSSSLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA PEAAGGPSVFLFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKPREEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKGQPREPQVYTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNRYTQKSLSLSPGK

Residues 1-126 of the third polypeptide chain of TRIDENT-D correspond to the VH domain of the optimized anti-gp 41 antibody 7B2GL (SEQ ID NO: 58). Residues 127-224 correspond to the IgG1 CH1 domain (SEQ ID NO: 3). Residue 225-239 corresponds to the hinge domain of IgG1(SEQ ID NO: 7). Residues 240-456 correspond to the CH2-CH3 domain of IgG1 "carrying the mortar" (SEQ ID NO: 50).

The fourth polypeptide chain of TRIDENT-D has the amino acid sequence of SEQ ID NO: 122: DIVMTQSPDS LAVSLGERAT INCKSSQTLL YSSNNRHSIA WYQQKPGQPPKLLIYWASMR LSGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCHQYSSHPPTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREAKVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKADYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

Residues 1-113 of the fourth polypeptide chain of TRIDENT-B correspond to the VL domain of anti-gp 41 antibody 7B2GL (SEQ ID NO: 57). Residues 114-220 correspond to the CL kappa domain (SEQ ID NO: 1).

H. Alternative molecules that multispecific bind gp41

As will be appreciated in light of the present disclosure, additional multispecific gp 41-binding molecules having the overall structure of any of the above-described exemplary molecules including the VL and VH domains of 7B2GL and including alternative ECM and/or HIV-1 antigen binding sites may be constructed by employing alternative VL and VH domains of ECM and/or HIV-1 antibodies in place of the VL and VH domains present in the above-described molecules. VL and VH domains from a number of alternative ECM and HIV-1 binding sites are provided herein, and additional VL and VH domains are known in the art. Similarly, as provided herein, alternative multispecific gp 41-binding molecules can likewise be constructed to incorporate alternative linkers and/or heterodimer promoting domains, particularly those provided herein.

Production method

The molecules of the present invention are most preferably produced by recombinant expression of a nucleic acid molecule encoding such a polypeptide, as is well known in the art.

Polypeptides Of The invention may conveniently be prepared using Solid Phase peptide Synthesis (Merrifield, B. (1986) "Solid Phase Synthesis," Science 232(4748):341 & 347; Houghten, R.A. (1985) "General Method For The Rapid Solid Phase Synthesis Of Large number Peptides: Specificity Of Antibody-Antibody Interaction At The Level Of Industrial Integrated Amino Acids," Proc. Natl. Acad. Sci. (U.S.A.)82(15):5131 & 5135; Ganesan, A. (2006) "Solid Phase Synthesis In two-First centre," Mini. Min. 6.1. 10 (3): 1.10).

Antibodies can be recombinantly produced and expressed using any method known in the art. Antibodies can be recombinantly produced by first isolating antibodies produced from a host animal, obtaining their DNA sequences, and using such DNA sequences to recombinantly express the antibodies in host cells (e.g., CHO cells). Another method that may be employed is the expression of antibody sequences in the milk of plants (e.g., tobacco) or transgenic animals. Suitable methods For Recombinant expression Of Antibodies In Plants or Milk have been disclosed (see, e.g., Peeters et al (2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19: 2756; Lonberg, N. et al (1995) "Human Antibodies From Transgenic Rice," int.Rev.Immunol 13: 65-93; And Pollock et al (1999) "Transgenic Milk As A Method For The Production Of Antibody Antibodies," J.Immunol.methods 231: 147-. Suitable methods for making antibody derivatives, e.g., humanized, single chain, etc., are known in the art and have been described above. In another alternative, Antibodies can be recombinantly produced By Phage Display Technology (see, e.g., U.S. Pat. Nos. 5,565,332, 5,580,717, 5,733,743, 6,265,150; and Winter, G. et al (1994) "Making Antibodies By phase Display Technology," Annu.Rev.Immunol.12.433-455).

Vectors containing a polynucleotide of interest (e.g., a polynucleotide encoding a polypeptide chain of a binding molecule of the invention) can be introduced into a host cell by any of a number of suitable means, including electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; bombardment of particles; lipofection; and infection (e.g., where the vector is an infectious agent, such as vaccinia virus). The choice of introducing a vector or polynucleotide will generally depend on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used for the purpose of expressing a polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells.

The invention includes polypeptides comprising the amino acid sequence of the binding molecules of the invention. The polypeptides of the invention can be prepared by procedures known in the art. The polypeptides may be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single polypeptides or fusion polypeptides), or by chemical synthesis. Polypeptides of antibodies, in particular shorter polypeptides of up to (up to) about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available.

The invention includes variants of the disclosed binding molecules, including functionally equivalent polypeptides that do not significantly affect the properties of such molecules, as well as variants with increased or decreased activity. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, the use of one or more deletions or additions of amino acids or chemical analogs that do not significantly or adversely alter functional activity. Amino acid residues that may be conservatively substituted for one another include, but are not limited to: glycine/alanine; serine/threonine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitution will be conservative, i.e., the substituted amino acid will have similar chemical properties of charge, size, etc. as the original amino acid. Such conservative substitutions are known in the art and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to fully redesigned regions, such as variable domains. Changes in the variable domains may alter binding affinity and/or specificity. Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. The modification may be used, for example, to attach a label for immunoassay, such as attaching a radioactive moiety for radioimmunoassay. Modified polypeptides are prepared using procedures established in the art and can be screened using standard assays known in the art.

In one embodiment, fusion polypeptides comprising a light chain, a heavy chain, or both a light chain and a heavy chain are provided. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises a VH domain and a VL domain of an antibody produced from a publicly deposited hybridoma. For the purposes of the present invention, an antibody fusion protein contains a polypeptide domain that enables the protein to immunospecifically bind gp41 and the ECM (e.g., CD3, CD16, etc.), and it contains another amino acid sequence not linked thereto in the native molecule, such as a heterologous or homologous sequence from another region (e.g., a domain that is deimmunized to bind albumin, a protein a recognition sequence, a peptide tag, etc.).

The invention specifically encompasses such binding molecules (e.g., antibodies, diabodies, trivalent binding molecules, etc.) conjugated to a diagnostic or therapeutic moiety. For diagnostic purposes, the binding molecules of the invention may be coupled to a detectable substance. Such binding molecules may be used as part of a clinical testing procedure for monitoring and/or prognosing the development or progression of a disease, such as determining the efficacy of a particular therapy. Examples of detectable substances include various enzymes (e.g., horseradish peroxidase, β -galactosidase, etc.), prosthetic groups (e.g., avidin/biotin), fluorescent substances (e.g., umbelliferone, fluorescein, or phycoerythrin), luminescent substances (e.g., luminol), bioluminescent substances (e.g., luciferase or luminogenic protein), radioactive substances (e.g., carbon 14, manganese 54, strontium 85, or zinc 65), positron emitting luminescent metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated to the binding molecule directly or indirectly through an intermediate (e.g., linker) using techniques known in the art.

For therapeutic purposes, the binding molecules of the invention may be conjugated to a therapeutic moiety, such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent, or a radioactive metal ion, e.g., an alpha-emitting agent. The cytotoxin or cytotoxic agent includes any agent that is harmful to cells, such as, for example, pseudomonas exotoxin, diphtheria toxin, botulinum toxins a through F, ricin, abrin, saporin, and cytotoxic fragments of such agents. Therapeutic agents include any agent that has a therapeutic effect to prophylactically or therapeutically treat discomfort. Such therapeutic agents may be chemotherapeutic agents, protein or polypeptide therapeutic agents, and include therapeutic agents that possess a desired biological activity and/or modify a given biological response. Examples of therapeutic agents include alkylating agents, angiogenesis inhibitors, antimitotic agents, hormonal therapy agents, and antibodies useful for treating cell proliferative disorders. The therapeutic moiety can be coupled or conjugated to the binding molecule directly or indirectly through an intermediate (e.g., linker) using techniques known in the art.

IX. use of the binding molecules of the invention

As discussed above, molecules capable of binding both gp41 and ECM are capable of mediating redirected cell killing of target cells expressing such gp41 on their cell surface (i.e., cells infected with a pathogen). Such molecules are useful for therapeutic purposes, for example in subjects suffering from an infection, in particular a latent HIV-1 infection. Thus, the binding molecules of the invention have the ability to treat diseases or conditions associated with expression of gp41, particularly latent HIV-1 infection, or characterized by expression of gp41, particularly latent HIV-1 infection. Thus, but not limited to, the binding molecules of the invention are useful in the treatment of HIV-1 infections, particularly latent HIV-1 infections.

In particular, the invention encompasses such methods, wherein the molecule capable of binding gp41 comprises a "binding epitope domain" capable of binding to an antibody of gp41 and also comprises a binding epitope domain capable of binding to the ECM (in particular, CD3 and/or CD16) on the surface of an immune effector cell so as to mediate redirected killing (e.g., by mediating redirected cell killing (e.g., redirected T-cell cytotoxicity)) of a target cell expressing gp 41.

In certain aspects, the invention provides the use of the gp 41-binding molecules of the invention, particularly multispecific gp 41-binding molecules such as, but not limited to, bispecific and trispecific molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent binding molecules, and the like), in methods of treating and preventing HIV-1 infection in an individual comprising administering to the individual in a pharmaceutically acceptable form a therapeutically effective amount of a composition comprising a gp 41-binding molecule of the invention. In certain embodiments, the molecules that bind gp41 bind different HIV-1 epitopes, preferably different epitopes present on the HIV-1 envelope.

Various molecules described herein that bind gp41 have utility, for example, in situations including, but not limited to:

i) in contemplated situations of known exposure to HIV-1 infection, the gp 41-binding molecules of the invention can be administered prophylactically (e.g., IV, topically, or intranasally) as microbicides,

ii) in the case of known or suspected exposure, such as occurs in the case of rape victims or commercial workers, or in any homosexual or heterosexual transmission without condom protection, the post-exposure prophylactic administration, e.g., IV or topical administration of gp 41-binding molecules of the invention, and

iii) in the case of acute HIV-1 infection (AHI), the gp 41-binding molecules of the invention can be administered as a treatment for AHI to control initial viral load, or for elimination of virus-infected CD 4T cells.

In accordance with the present invention, the gp 41-binding molecule of the present invention can be administered prior to or within about 48 hours of contacting the subject or subject's immune system/cell with HIV-1. Administration over this time frame maximizes inhibition of HIV infection of susceptible cells in the subject.

In addition, various forms of gp 41-binding molecules of the invention can be administered to chronically or acutely infected HIV-1 subjects and used to kill remaining virus-infected cells, since these gp 41-binding molecules bind to the surface of virus-infected cells and are capable of mediating redirected cell killing of such infected cells.

In certain embodiments, a gp 41-binding molecule of the invention can be administered in combination with a latent activator, in order to activate a latent pool of HIV-infected cells that may be present in a subject. It is expected that by activating the HIV-1DNA of the latent provirus in resting cells, previously inactive cells will start to produce new virus and will be recognized and eliminated by the immune system that has been amplified by the gp 41-binding molecule of the present invention. Non-limiting examples of latency activating agents are HDAC inhibitors, e.g., vorinostat, romidepsin, panobinostat, disulfiram, JQ1, bryoid, PMA, ionomycin, or any combination thereof. See Bullen et al Nature Medicine 20, 425-.

In certain embodiments, the gp 41-binding molecules of the invention can be administered in combination with an antiretroviral agent.

In particular embodiments, the molecule capable of binding gp41 and ECM is a bispecific antibody,Or

In a specific embodiment, the molecule capable of binding gp41 and ECM is a bispecific diabody.

In a specific embodiment, the molecule capable of binding gp41 and the ECM is a trivalent binding molecule.

As used herein, the term: "providing therapy" and "treatment" refer to any administration of a composition in association with any indication of a favorable or desired outcome, including but not limited to any clinical outcome, such as reduction of symptoms caused by disease; alleviating symptoms of infection (e.g., viral load, fever, pain, sepsis, etc.); reduction of symptoms caused by disease; an increase in the quality of life of the recipient subject; providing a reduction in the dosage of the other drug to treat the disease in the subject; enhancement of another drug effect such as targeted and/or internalization; delay in disease progression; and/or prolongation of survival of the subject.

Subjects for treatment include animals, most preferably mammalian species, such as non-primates (e.g., cows, horses, cats, dogs, rodents, etc.) or primates (e.g., monkeys such as cynomolgus monkeys, humans, etc.). In a preferred embodiment, the subject is a human.

Exemplary maladies treatable by various embodiments of the present invention include, but are not limited to, HIV-1 infection (particularly latent HIV-1 infection associated with expression of gp41 bound by molecules capable of mediating redirected cell killing). In various embodiments, the invention encompasses methods or compositions for treating, preventing, or managing a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of a binding molecule of the invention. Such molecules are particularly useful for reducing HIV-1 load or eliminating HIV-infected cells. While not wishing to be bound by a particular mechanism of action, such molecules may mediate effector functions on target cells, promote activation of the immune system against target cells, crosslink cell surface antigens and/or receptors on target cells, and enhance apoptosis or negative growth regulatory signals, or a combination thereof, resulting in clearance and/or reduced number of target cells.

X. pharmaceutical composition

The invention encompasses compositions comprising gp 41-binding molecules of the invention. The compositions of the invention include bulk drug compositions (e.g., impure or non-sterile compositions) that can be used to manufacture pharmaceutical compositions and pharmaceutical compositions that can be used in unit dosage form formulations (i.e., compositions suitable for administration to a subject). Such compositions include a prophylactically or therapeutically effective amount of a molecule (i.e., gp41 x ECM-binding molecule) that is capable of binding gp41 and is also capable of binding to the ECM so as to be capable of mediating redirected killing of a target cell (e.g., HIV-infected cells, etc.), or a combination of such an agent and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of a binding molecule of the invention and a pharmaceutically acceptable carrier. In a preferred aspect, such compositions are substantially pure (i.e., substantially free of materials that limit their effectiveness or produce undesirable side effects).

Various formulations of such compositions are useful for administration. In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries, which are well known in the art and which are relatively inert substances which facilitate the administration of pharmacologically active substances or the processing of the active compounds into preparations which can be pharmaceutically used for delivery to the site of action. For example, an excipient may impart a shape (form) or consistency (consistency) or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying the osmotic pressure, encapsulating agents, buffers, and skin penetration enhancers.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered. Generally, the ingredients of the compositions of the present invention are provided separately or mixed together in unit dosage form, for example as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet (sachette) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed from an infusion bottle containing sterile pharmaceutical grade water or physiological saline (saline). When the composition is administered by injection, an ampoule of sterile water or physiological saline for injection may be provided so that the ingredients may be mixed prior to administration.

The invention also provides a pharmaceutical pack (pack) or kit comprising one or more containers filled with a gp 41-binding molecule of the invention alone or with gp 41-binding molecule of the invention and such a pharmaceutically acceptable carrier. In addition, one or more other prophylactic or therapeutic agents for treating a disease may also be included in a pharmaceutical package or kit. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the invention. Optionally associated with such a container may be a notice (notice) in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The present invention provides kits useful in the above methods. Kits may include any of the binding molecules of the invention. The kit may further comprise in one or more containers one or more other prophylactic and/or therapeutic agents for treating cancer.

Xi. method of administration

The compositions of the invention can be provided for the treatment, prevention, and amelioration of one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of a pharmaceutical composition comprising a gp 41-binding molecule of the invention. In a preferred aspect, such compositions are substantially pure (i.e., substantially free of materials that limit their effectiveness or produce undesirable side effects). In particular embodiments, the subject is an animal, preferably a mammal, such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey, such as cynomolgus monkey, human, etc.). In a preferred embodiment, the subject is a human.

Methods of administering the gp 41-binding molecules of the invention or compositions comprising such gp 41-binding molecules include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In specific embodiments, the binding molecules of the invention are administered intramuscularly, intravenously or subcutaneously. The compositions can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous protective layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local.

The invention also provides that a preparation of the gp 41-binding molecule of the invention is packaged in a hermetically sealed container, such as an ampoule or sachet, which indicates the amount of the molecule. In one embodiment, such gp 41-binding molecules are provided as a dry sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to an appropriate concentration for administration to a subject. Preferably, the gp 41-binding molecules of the present invention are provided as sterile lyophilized powders in hermetically sealed containers.

Lyophilized formulations of gp 41-binding molecules of the invention should be stored in their initial container between 2 ℃ and 8 ℃ and the molecule should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours or within 1 hour after reconstitution. In an alternative embodiment, such molecules are provided in liquid form in hermetically sealed containers that indicate the amount and concentration of the molecules, fusion proteins or conjugate molecules. Preferably, such binding molecules when provided in liquid form are provided in a hermetically sealed container.

The amount of such a formulation of the invention effective to treat, prevent or ameliorate one or more symptoms associated with the disorder can be determined by standard clinical techniques. The precise dosage employed in the formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and the circumstances of each recipient subject. Effective doses can be extrapolated from dose response curves derived from in vitro or animal model test systems.

As used herein, an "effective amount" of a pharmaceutical composition is an amount sufficient to achieve a beneficial or desired result, including, but not limited to, a clinical result, such as alleviating symptoms caused by a disease, alleviating symptoms of an infection (e.g., viral load, fever, pain, sepsis, etc.), thereby improving the quality of life of those suffering from a disease, reducing the dosage of other drugs needed to treat a disease, enhancing the effect of another drug treatment, such as targeting and/or internalization, delaying the progression of a disease, and/or prolonging survival of an individual. When applied to a single active ingredient when administered alone, the term refers to that individual ingredient. When applied to a combination, the term refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, sequentially or simultaneously.

An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is a sufficient amount: to kill and/or reduce the proliferation of HIV-1 infected cells, either directly or indirectly; or reducing the proliferation (or effect) of the HIV-1 virus and reducing and/or delaying the progression of an HIV-mediated disease. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without the incorporation of another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more chemotherapeutic agents, and if the desired result can be achieved or achieved in combination with one or more other agents, it is contemplated that a single agent may be administered in an effective amount. The optimal range for determining the effective amount of each component is within the skill of one in the art, although individual requirements vary.

For binding molecules encompassed by the present invention, the dosage administered to the recipient subject is preferably determined based on the weight (kg) of the recipient subject. For binding molecules encompassed by the present invention, the dose administered to a recipient subject is typically from about 0.01 μ g/kg subject body weight to about 30mg/kg subject body weight or higher.

The dose and frequency of administration of the binding molecules of the invention may be reduced or altered by modifications such as, for example, lipidation enhancing molecules absorption and tissue penetration.

The dosage of a binding molecule of the invention administered to a recipient subject as a single agent therapy can be calculated. Alternatively, the molecule may be used in combination with other therapeutic compositions and the dose administered to the recipient subject is less than the dose used when the molecule is used as a single agent therapy.

The pharmaceutical compositions of the present invention may be administered topically to the area in need of treatment; this may be achieved, for example, but not limited to, by local infusion, by injection, or by implants which are porous, non-porous, or gelatinous materials including membranes, such asA film or a fiber. Preferably, when applying the molecules of the invention, care must be taken to use materials that do not adsorb the molecules.

The compositions Of the invention may be delivered in vesicles, particularly liposomes (see Langer (1990) "New Methods Of Drug Delivery," Science 249: 1527-; treat et al, in Liposomes in the Therapy of infection Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, supra, pp.317-327).

Treatment of a subject with a therapeutically or prophylactically effective amount of a binding molecule of the invention may comprise a single treatment, or preferably may comprise a series of treatments. In a preferred example, the subject is treated with the pharmaceutical composition of the invention for between about 1and 10 weeks, preferably between 2 and 8 weeks, more preferably between about 3 and 7 weeks, even more preferably for about 4 weeks, 5 weeks or 6 weeks. The pharmaceutical compositions of the present invention may be administered once daily, wherein such administration is performed once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, once a year, or the like. Alternatively, the pharmaceutical composition of the present invention may be administered twice a day, wherein such administration is performed once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, once a year, or the like. Alternatively, the pharmaceutical composition of the present invention may be administered three times a day, wherein such administration is performed once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, once a year, or the like. It will also be appreciated that the effective dose of the molecule for treatment may be increased or decreased over the course of a particular treatment.

Examples

Having generally described this invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless otherwise specified.

Example 1

Optimization

An anti-HIV-1 Env (GP41 protein) Antibody designated "7B 2 IgG" (GenBank accession number AFQ 31503; Buchacher, A. et al (1994) "Generation Of Human Monoclonal Antibodies Against HIV-1 Proteins; Electrofusion And animal Blood Transformation For Peripheral Blood Lymphocyte activation," AIDS Res.hum.Retroviruses 10(4):359 369; Shen, R2010 (GP 41-Specific Antibodies Blocks-Free HIV-1Type 1 cells Through Human recovery modified Antibody Model, "J.7. immune Antibody Model 3655) contains a number Of Human IgG residues in its framework region 3655. The presence of such rare non-germline residues can be immunogenic and result in the production of anti-drug-antibodies when repeatedly administered to a recipient subject. To reduce or even eliminate the immunogenic potential of these domains, the 7B2 variable region was optimized by introducing mutations into its framework regions to replace such rare amino acid residues with germline amino acid residues.

As shown in fig. 7A-7B, nine (9) mutations were introduced into the VL framework regions (fig. 7A) and seventeen (17) amino acid substitutions were introduced into the VH framework regions (fig. 7B) to produce fully germlined VL and VH domains designated "7B 2GL VL" and "7B 2GL VH", respectively. The resulting VL/VH domains are collectively referred to as "7B 2 GL".

The amino acid sequences of the VL domains of antibody 7B2 and antibody 7B2GL have been presented above (SEQ ID NO:55 and SEQ ID NO:57, respectively). The amino acid sequences of the VL domain of the framework 1-3 donor (IGKV4-1) and the framework 4 region of the framework 4 donor (IGKJ1-01) are:

IGKV4-1(SEQ ID NO:123)：

DIVMTQSPDS LAVSLGERAT INCKSSQSVL YSSNNKNYLA WYQQKPGQPP KLLIYWASTR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQQYYST P

and IGKJ1-01(SEQ ID NO: 124):

FGQGTKVEIK。

the amino acid sequences of the VH domains of antibody 7B2 and antibody 7B2GL have been presented above (SEQ ID NO:56 and SEQ ID NO:58, respectively). The amino acid sequences of the VH domain of the framework 1-3 donor (IGVH3-11) and the framework 4 region of the framework 4 donor (IGHJ4-01) were:

IGVH3-11(SEQ ID NO:125)：

QVQLVESGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQAPGKGLEWVSY ISSSSSYTNY ADSVKGRFTI SRDNAKNSLY LQMNSLRAEDTAVYYCAR

and IGHJ4-01(SEQ ID NO: 126):

WGQGTLVTVSS

binding of IgG comprising fully germlined variable regions (designated "7B 2GL IgG") was detected by Attana Cell a200 QCM. Briefly, the HIV-1JRFL gp140 recombinant protein (HIV-1Env protein minus transmembrane and C-terminal amino acid residues, but retaining the gp41 epitope recognized by 7B 2) was independently captured at 25nM, 50nM and 100nM at 7B2 IgG (FIG. 8A) or 7B2GL IgG (FIG. 8B) on an Attana sensor chip surface-coated with anti-human IgG Fc. For the analysis of kinetic parameters (ka, KD), the 1:1 binding was chosen as a suitable model since it is the mathematically least manipulated model. However, because JRFL gp140 is a mixture of monomers (20-30%), dimers (40%), and trimers (30%), the model does not completely describe the interaction. Accordingly, the kinetic parameters (ka, KD, using a 1:1 binding fit) provided in table 8 were used primarily for the purpose of directly comparing the Ag binding activity of the 7B2 and 7B2GL constructs to each other. Although many Of The alterations are located in The regions known to be important for antigen binding (e.g., VH domain Kabat positions: 30, 48, 49 and 79, and VL domain Kabat position 48) (Foote and Winter (1992) "Antibody Framework reactions influencing The compatibility Of The Hypervariable Loops," J.Mol.biol.224:487 499) and generally do not alter (see, e.g., Chromikova et al (2015) "Introduction Of plasmid Of The Stability Of Anti-HIV mAb2G12-IgM," Biochim.Biophys.acta 1854: 1536) -1544) The epitopes (Vernier) regions, these studies show that all 26 Germline mutations are well-tolerated and that fully Germline antibodies display nearly The same kinetic binding as wild type B2 Vernier antibodies.

Example 2

HIV x CD3 binding molecules

Two HIV x CD3 binding molecules capable of binding to the HIV Env gp41 protein and the exemplary immune effector cell target, CD3, were generated. In particular, two bispecific diabodies (designated "DART-1" and "DART-a") comprising 7B2 or 7B2GL variable domains and having three polypeptide chains were produced, each comprising a CD3mAb 1(D65G) variable domain. DART-1 and DART-A are Fc domain-containing bispecific diabodies with the overall structure shown in FIG. 4A that are capable of binding to HIV and CD 3. The domains and amino acid sequences of these exemplary molecules are described in detail above.

The biological activity of DART-1 and DART-A was mutexamined in a number of tests. DART-1 and DART-A were evaluated by flow cytometry for their ability to bind gp41 mutexpressed on the surface of HEK/D371 cells. Briefly, gp 140-mutexpressing HEK293/D371 cells (doxycycline-inducible mutexpressed HIV-1CM244 (subtype AE) gp140 envelope, obtained from dr. john kappa, (University of Alabama, Birmingham) after pre-incubation with 1 μ g/mL doxycycline for 24 hours) were incubated with sequential dilutions (10 μ g/mL to 0.0024 μ g/mL) of DART-1, DART-a, or RSV x CD3 negative controls. After washing, cells were incubated with Alexa 488-anti-human IgG1 Fc secondary antibody and analyzed by flow cytometry (e.g., MFI plotted using Guava EasyCyte 8ht (millipore) and by GraphPad Prism 7). As shown in FIG. 9, DART-1 and DART-A bound HEK293/D371 cells mutexpressing gp140 in a similar dose-dependent manner.

The ability of DART-1 and DART-A to activate T cells was evaluated using the Jurkat T cell reporter test (Promega). Briefly, Jurkat IL2 Luc2P reporter cells were co-cultured with HEK293/D371 cells mutexpressing gp140 (20:1) in the presence of DART-1, DART-A or RSV x CD3 negative controls at serial dilutions (10. mu.g/mL-0.0024. mu.g/mL) and used ONE-GLO^TMThe luciferase assay system (Promega) measures luminescence. As shown in FIG. 10, DART-1 and DART-A mutexhibited comparable T-cell activation activity.

The ability of DART-1 and DART-a to mediate T cell redirected target cell killing was assessed using CTL assays. Briefly, Pan T cells are isolated from healthy human PBMCs (e.g., usingUntouched^TMHuman T cell kit (Invitrogen)). With an effector: target (E: T) ratio of 10:1, or with human T cells in other ways that alter E: T ratio as indicated, gp 140-mutexpressing HEK293/D371(1-4x 10) was treated with serial dilutions of DART-1, DART-a or RSV x CD3 negative controls⁵cells/mL) and 5% CO at 37 deg.C₂Incubate for 24 hours. Release by Lactate Dehydrogenase (LDH) (e.g., using CytoTox)Non-radioactive cytotoxicity test, Promega) to measure cytotoxicity. As shown in figure 11, DART-1 and DART-a mutexhibited comparable T cell redirecting target cell killing activity.

The results of these studies indicate that the introduction of many substitutions into the VL and VH domains of 7B2 to produce an optimized fully germlined 7B2GL domain did not affect gp140 binding or biological activity in the above assays.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Sequence listing

<110> Macro Gene Co Ltd

Lam, Chia-Yang

Diedrich, Gundo

<120> optimized gp 41-binding molecules and uses thereof

<130> 1301.0155PCT

<150> US 62/673,462

<151> 2018-05-18

<160> 131

<170> PatentIn version 3.5

<210> 1

<211> 107

<212> PRT

<213> Intelligent people

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<221> MISC_FEATURE

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<223> human IgG CL kappa Domain

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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

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Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 2

<211> 104

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(104)

<223> human IgG CL lambda Domain

<400> 2

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

50 55 60

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

65 70 75 80

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

85 90 95

Thr Val Ala Pro Thr Glu Cys Ser

100

<210> 3

<211> 98

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(98)

<223> human IgG1 CH1 Domain

<400> 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val

<210> 4

<211> 98

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(98)

<223> human IgG2 CH1 Domain

<400> 4

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val

<210> 5

<211> 98

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(98)

<223> human IgG3 CH1 Domain

<400> 5

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val

<210> 6

<211> 98

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(98)

<223> human IgG4 CH1 Domain

<400> 6

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val

<210> 7

<211> 15

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(15)

<223> human IgG1 hinge domain

<400> 7

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 8

<211> 12

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> human IgG2 hinge domain

<400> 8

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> 9

<211> 62

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(62)

<223> human IgG3 hinge domain

<400> 9

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

20 25 30

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu

35 40 45

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

50 55 60

<210> 10

<211> 12

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> human IgG4 hinge domain

<400> 10

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> IgG4 hinge domain comprising a stable S228P substitution

<400> 11

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 12

<211> 217

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(217)

<223> exemplary human IgG1 CH2-CH3 Domain

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 12

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 13

<211> 216

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(216)

<223> exemplary human IgG2 CH2-CH3 Domain

<220>

<221> MISC_FEATURE

<222> (216)..(216)

<223> Xaa is lysine (K) or absent

<400> 13

Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

1 5 10 15

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

20 25 30

Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

35 40 45

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

50 55 60

Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln

65 70 75 80

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

85 90 95

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro

100 105 110

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

115 120 125

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

130 135 140

Asp Ile Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

145 150 155 160

Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

165 170 175

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

180 185 190

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

195 200 205

Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 14

<211> 217

<212> PRT

<213> Intelligent people

<220>

<221> 'MISC_FEATURE

<222> (1)..(217)

<223> exemplary human IgG3 CH2-CH3 Domain

<220>

<221> 'MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 14

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 15

<211> 217

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(217)

<223> exemplary human IgG4 CH2-CH3 Domain

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 15

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> preferred Interval peptide (linker 1)

<400> 16

Gly Gly Gly Ser Gly Gly Gly Gly

1 5

<210> 17

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> preferred cysteine-containing spacer peptide (linker 2)

<400> 17

Gly Gly Cys Gly Gly Gly

1 5

<210> 18

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 18

Gly Gly Gly Ser

1

<210> 19

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 19

Leu Gly Gly Gly Ser Gly

1 5

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 20

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

1 5 10

<210> 21

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 21

Ala Ser Thr Lys Gly

1 5

<210> 22

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 22

Leu Glu Pro Lys Ser Ser

1 5

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> optional linker 2 spacer peptide without cysteine

<400> 23

Ala Pro Ser Ser Ser

1 5

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> heterodimer promoting Domain

<400> 24

Gly Val Glu Pro Lys Ser Cys

1 5

<210> 25

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> heterodimer promoting Domain

<400> 25

Val Glu Pro Lys Ser Cys

1 5

<210> 26

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> heterodimer promoting Domain

<400> 26

Ala Glu Pro Lys Ser Cys

1 5

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> heterodimer promoting Domain

<400> 27

Gly Phe Asn Arg Gly Glu Cys

1 5

<210> 28

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> heterodimer promoting Domain

<400> 28

Phe Asn Arg Gly Glu Cys

1 5

<210> 29

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> "E-helix" heterodimer promoting Domain

<400> 29

Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val

1 5 10 15

Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

20 25

<210> 30

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> "K-helix" heterodimer promoting Domain

<400> 30

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val

1 5 10 15

Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

20 25

<210> 31

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> cysteine-containing "E-helix" heterodimer promoting domain

<400> 31

Glu Val Ala Ala Cys Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val

1 5 10 15

Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

20 25

<210> 32

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> cysteine-containing "K-helix" heterodimer promoting domain

<400> 32

Lys Val Ala Ala Cys Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val

1 5 10 15

Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu

20 25

<210> 33

<211> 46

<212> PRT

<213> Streptococcus dysgalactiae

<220>

<221> MISC_FEATURE

<222> (1)..(46)

<223> Albumin binding Domain 3 of protein G of Streptococcus strain G148 (ABD3)

<400> 33

Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly

1 5 10 15

Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu

20 25 30

Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro

35 40 45

<210> 34

<211> 46

<212> PRT

<213> Artificial sequence

<220>

<223> N66D/T70S/V71A variant of protein G of Streptococcus strain G148 deimmunized Albumin binding Domain 3(ABD3)

<400> 34

Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly

1 5 10 15

Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu

20 25 30

Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro

35 40 45

<210> 35

<211> 46

<212> PRT

<213> Artificial sequence

<220>

<223> L64A/I65A/D79A variant of protein G of Streptococcus strain G148 deimmunized Albumin binding Domain 3(ABD3)

<400> 35

Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly

1 5 10 15

Val Ser Asp Tyr Tyr Lys Asn Ala Ala Asn Asn Ala Lys Thr Val Glu

20 25 30

Gly Val Lys Ala Leu Ile Ala Glu Ile Leu Ala Ala Leu Pro

35 40 45

<210> 36

<211> 46

<212> PRT

<213> Artificial sequence

<220>

<223> N66S/T70S/D79A variant of protein G of Streptococcus strain G148 deimmunized Albumin binding Domain 3(ABD3)

<400> 36

Leu Ala Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly

1 5 10 15

Val Ser Asp Tyr Tyr Lys Asn Leu Ile Ser Asn Ala Lys Ser Val Glu

20 25 30

Gly Val Lys Ala Leu Ile Ala Glu Ile Leu Ala Ala Leu Pro

35 40 45

<210> 37

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Interval peptide (linker)

<400> 37

Ala Pro Ser Ser Ser Pro Met Glu

1 5

<210> 38

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Interval peptide (linker)

<400> 38

Val Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 39

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Interval peptide (linker)

<400> 39

Leu Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Interval peptide (linker)

<400> 40

Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> 41

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> scFv spacer peptide linker

<400> 41

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 42

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> "Long" Interval peptide linker

<400> 42

Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

1 5 10

<210> 43

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> alternative intervening spacer peptide (linker)

<400> 43

Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> 44

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> alternative intervening spacer peptide (linker)

<400> 44

Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 45

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary human IgG1 CH2-CH3 domain with L234A/L235A substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 45

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 46

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary human IgG1 CH2-CH3 domain with M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 46

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 47

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary human IgG1 CH2-CH3 Domain with L234A/L235A/M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 47

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 48

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary "knob-carrying" human IgG1 CH2-CH3 domain with L234A/L235A substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 48

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 49

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary "knob-carrying" human IgG1 CH2-CH3 domain with L234A/L235A/M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 49

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 50

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> an exemplary "mortar carrying" human IgG1 CH2-CH3 domain with L234A/L235A substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> XAA is lysine (K) or absent

<400> 50

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 51

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> human IgG1 CH2-CH3 domain with L234A/L235A/M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 51

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Xaa

210 215

<210> 52

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary human IgG4 CH2-CH3 domain with M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 52

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215

<210> 53

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> exemplary "knob-carrying" human IgG4 CH2-CH3 domain with M252Y/S254T/T256E substitutions

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 53

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215

<210> 54

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> human IgG4 CH2-CH3 Domain with M252Y/S254T/T256E substitution exemplary "carrying the mortar

<220>

<221> MISC_FEATURE

<222> (217)..(217)

<223> Xaa is lysine (K) or absent

<400> 54

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Xaa

210 215

<210> 55

<211> 113

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(113)

<223> VL domain of anti-HIV Env human IgG1 antibody 7B2

<400> 55

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Ile His Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Arg Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Leu Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Asn Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly His Gly Thr Arg Val Glu Ile

100 105 110

Lys

<210> 56

<211> 126

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(126)

<223> VH Domain of anti-HIV Env human IgG1 antibody 7B2

<400> 56

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Phe Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Thr Glu Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Ala Tyr Ile Ser Lys Asn Gly Glu Tyr Ser Lys Tyr Ser Pro Ser Ser

50 55 60

Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Phe

65 70 75 80

Leu Gln Leu Asp Arg Leu Ser Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Gly Leu Thr Tyr Phe Ser Glu Leu Leu Gln Tyr Ile

100 105 110

Phe Asp Leu Trp Gly Gln Gly Ala Arg Val Thr Val Ser Ser

115 120 125

<210> 57

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of optimized anti-HIV Env human IgG1 antibody 7B2GL

<400> 57

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 58

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of optimized anti-HIV Env human IgG1 antibody 7B2GL

<400> 58

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Lys Asn Gly Glu Tyr Ser Lys Tyr Ser Pro Ser Ser

50 55 60

Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Gly Leu Thr Tyr Phe Ser Glu Leu Leu Gln Tyr Ile

100 105 110

Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 59

<211> 118

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(118)

<223> VH Domain of anti-human CD2 mAb Lo-CD2a

<400> 59

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Gln Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu Tyr

20 25 30

Tyr Met Tyr Trp Val Lys Gln Arg Pro Lys Gln Gly Leu Glu Leu Val

35 40 45

Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe

50 55 60

Lys Lys Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 60

<211> 112

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(112)

<223> VL domain of anti-human CD2 mAb Lo-CD2a

<400> 60

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Leu Leu Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Thr Gly Gln Ser

35 40 45

Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Met Gln Phe

85 90 95

Thr His Tyr Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 61

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD3mAb1

<400> 61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 62

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD3mAb1

<400> 62

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 63

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD3mAb 1(D65G)

<400> 63

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 64

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD3mAb1 Low

<400> 64

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Thr Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 65

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> fast VH Domain of humanized anti-human CD3mAb1

<400> 65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Lys Asn Phe Gly Asn Ser Tyr Val Thr Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 66

<211> 119

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(119)

<223> VH Domain of anti-human CD3 antibody OKT3

<400> 66

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 67

<211> 107

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(107)

<223> VL domain of anti-human CD3 antibody OKT3

<400> 67

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg

100 105

<210> 68

<211> 120

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(120)

<223> VH Domain of anti-human CD8 antibody OKT8

<400> 68

Gln Val Gln Leu Leu Glu Ser Gly Pro Glu Leu Leu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Thr Gly Gly Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Asn Lys Ala Thr Leu Thr Val Asp Ser Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Phe Arg Tyr Thr Tyr Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 69

<211> 112

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(112)

<223> VL domain of anti-human CD8 antibody OKT8

<400> 69

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Asp Asn Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Val Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 70

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD8 antibody TRX2

<400> 70

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 71

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD8 antibody TRX2

<400> 71

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asp Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 72

<211> 118

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(118)

<223> VH Domain of anti-human CD16 antibody 3G8

<400> 72

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Arg Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 73

<211> 111

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(111)

<223> VL domain of anti-human CD16 antibody 3G8

<400> 73

Asp Thr Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 74

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD16 antibody h3G8

<400> 74

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 75

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD16 antibody h3G8

<400> 75

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 76

<211> 117

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(117)

<223> VH Domain of anti-human CD16 antibody A9

<400> 76

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Val Thr Ala Asp Thr Ser Ser Arg Thr Ala Tyr

65 70 75 80

Val Gln Val Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Ala Ser Trp Tyr Phe Asp Val Trp Gly Ala Arg Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 77

<211> 111

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(111)

<223> VL domain of anti-human CD16 antibody A9

<400> 77

Asp Ile Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro

1 5 10 15

Gly Glu Thr Val Thr Leu Thr Cys Arg Ser Asn Thr Gly Thr Val Thr

20 25 30

Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe

35 40 45

Thr Gly Leu Ile Gly His Thr Asn Asn Arg Ala Pro Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr

65 70 75 80

Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr

85 90 95

Asn Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 78

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 78

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ala Tyr Tyr Tyr Asp Phe Ala Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 79

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> first exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 79

Ser Tyr Glu Leu Met Gln Pro Pro Ser Val Ser Val Ser Ser Gly Gln

1 5 10 15

Thr Ala Ser Ile Pro Cys Ser Gly Asp Lys Leu Glu Glu Lys Tyr Val

20 25 30

Ser Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 80

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> second exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 80

Ser Tyr Glu Leu Thr Gln Pro Leu Ser Glu Ser Val Ala Gln Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Glu Ser Arg Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Met Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Ala Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Thr Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 81

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> third exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 81

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ala Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Phe Tyr Cys Gln Val Trp Asp Asn Tyr Ile Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 82

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> fourth exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 82

Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Pro Cys Glu Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Arg Gln Lys Pro Gly Gln Val Pro Val Leu Val Met Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 83

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> fifth exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 83

Gln Pro Val Leu Thr Gln Pro Leu Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Arg Asp Ser Ser Arg Pro Ser Gly Ile Pro Glu Arg Leu Ser Gly Ser

50 55 60

Asn Ser Gly Asp Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asp Tyr Ile Val Val

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 84

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> sixth exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 84

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Thr Pro Gly Gln

1 5 10 15

Thr Ala Thr Ile Thr Cys Gly Ala Asn Asp Ile Gly Lys Arg Asn Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 85

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> seventh exemplary VL domain of anti-human CD16A antibody of U.S. Pat. No. 9,035,026

<400> 85

Gln Pro Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Thr Ile Ser Cys Gly Gly His Asn Ile Gly Ser Lys Asn Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Asn Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asn Tyr Ser Val Leu

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 86

<211> 120

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(120)

<223> VH Domain of anti-human TCR antibody BMA 031

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys

85 90 95

Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 87

<211> 106

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(106)

<223> VL domain of anti-human TCR antibody BMA 031

<400> 87

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Thr Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 88

<211> 118

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(118)

<223> VH Domain of human anti-human NKG2D receptor antibody KYK-1.0

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Lys Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Phe Gly Tyr Tyr Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 89

<211> 108

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(108)

<223> VL domain of human anti-human NKG2D receptor antibody KYK-1.0

<400> 89

Gln Pro Val Leu Thr Gln Pro Ser Ser Val Ser Val Ala Pro Gly Glu

1 5 10 15

Thr Ala Arg Ile Pro Cys Gly Gly Asp Asp Ile Glu Thr Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Asp Asp Asp Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Phe Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Ser Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asp Asn Asn Asp Glu

85 90 95

Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu

100 105

<210> 90

<211> 121

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(121)

<223> VH Domain of human anti-human NKG2D receptor antibody KYK-2.0

<400> 90

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 91

<211> 110

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(110)

<223> VL domain of human anti-human NKG2D receptor antibody KYK-2.0

<400> 91

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 92

<211> 117

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(117)

<223> VH Domain of anti-human NKp46 antibody BAB281

<400> 92

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Gln Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asp Tyr Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 93

<211> 108

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(108)

<223> VL domain of anti-human NKp46 antibody BAB281

<400> 93

Asp Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val

1 5 10 15

Gly Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr

50 55 60

Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln

65 70 75 80

Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 94

<211> 116

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(116)

<223> VH Domain of anti-human NKp46 antibody NKp46-3

<400> 94

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ser Pro Asn Ile Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 95

<211> 107

<212> PRT

<213> mouse

<220>

<221> MISC_FEATURE

<222> (1)..(107)

<223> VL domain of anti-human NKp46 antibody NKp46-3

<400> 95

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 96

<211> 123

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(123)

<223> VH Domain of human anti-HIV-1 Env gp120 antibody A32

<400> 96

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Ser Cys Thr Val Ser Gly Gly Ser Ser Ser Ser Gly

20 25 30

Ala His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Thr Ile Ser Gln His Thr Ser Glu Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Val Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Thr Arg Leu Arg Thr Leu Arg Asn Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 97

<211> 110

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(110)

<223> VL domain of human anti-HIV-1 Env gp120 antibody A32

<400> 97

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Ser Glu Val Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Ser Ser Tyr Thr Asp Ile

85 90 95

His Asn Phe Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 98

<211> 132

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-HIV-1 Env gp120 antibody 10-1074

<400> 98

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn Tyr

20 25 30

Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn

50 55 60

Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu

65 70 75 80

Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu

100 105 110

Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val

115 120 125

Thr Val Ser Ser

130

<210> 99

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-HIV-1 Env gp120 antibody 10-1074

<400> 99

Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr Ala Arg Ile

1 5 10 15

Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln

20 25 30

His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn Asn Gln Asp

35 40 45

Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp Ile Asn

50 55 60

Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser Gly Phe Ser

85 90 95

Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu

100 105

<210> 100

<211> 123

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(123)

<223> VH Domain of humanized anti-HIV-1 Env gp120 antibody 3BNC117

<400> 100

Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr

20 25 30

Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val

35 40 45

Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe

50 55 60

Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr

65 70 75 80

Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala

85 90 95

Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val

100 105 110

Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 101

<211> 99

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(99)

<223> VL domain of humanized anti-HIV-1 Env gp120 antibody 3BNC117

<400> 101

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr

20 25 30

Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser

35 40 45

Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly

50 55 60

Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala

65 70 75 80

Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu

85 90 95

Asp Leu Lys

<210> 102

<211> 132

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(132)

<223> VH Domain of humanized anti-HIV-1 Env gp120 antibody PGT121

<400> 102

Gln Met Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Ala Ser Ile Ser Asp Ser

20 25 30

Tyr Trp Ser Trp Ile Arg Arg Ser Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Val His Lys Ser Gly Asp Thr Asn Tyr Ser Pro Ser Leu Lys

50 55 60

Ser Arg Val Asn Leu Ser Leu Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Ser Leu Val Ala Ala Thr Ala Ala Asp Ser Gly Lys Tyr Tyr Cys Ala

85 90 95

Arg Thr Leu His Gly Arg Arg Ile Tyr Gly Ile Val Ala Phe Asn Glu

100 105 110

Trp Phe Thr Tyr Phe Tyr Met Asp Val Trp Gly Asn Gly Thr Gln Val

115 120 125

Thr Val Ser Ser

130

<210> 103

<211> 105

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(105)

<223> VL domain of PGT121 of humanized anti-HIV-1 Env gp120 antibody

<400> 103

Ser Asp Ile Ser Val Ala Pro Gly Glu Thr Ala Arg Ile Ser Cys Gly

1 5 10 15

Glu Lys Ser Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln His Arg Ala

20 25 30

Gly Gln Ala Pro Ser Leu Ile Ile Tyr Asn Asn Gln Asp Arg Pro Ser

35 40 45

Gly Ile Pro Glu Arg Phe Ser Gly Ser Pro Asp Ser Pro Phe Gly Thr

50 55 60

Thr Ala Thr Leu Thr Ile Thr Ser Val Glu Ala Gly Asp Glu Ala Asp

65 70 75 80

Tyr Tyr Cys His Ile Trp Asp Ser Arg Val Pro Thr Lys Trp Val Phe

85 90 95

Gly Gly Gly Thr Thr Leu Thr Val Leu

100 105

<210> 104

<211> 140

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(140)

<223> VH Domain of humanized anti-HIV-1 Env gp120 antibody PGT145

<400> 104

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asn Ser Phe Ser Asn His

20 25 30

Asp Val His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Ser His Glu Gly Asp Lys Thr Gly Leu Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Ser Gly Ala Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Arg Gly Leu Thr Ala Asp Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Leu Thr Gly Ser Lys His Arg Leu Arg Asp Tyr Phe Leu Tyr Asn Glu

100 105 110

Tyr Gly Pro Asn Tyr Glu Glu Trp Gly Asp Tyr Leu Ala Thr Leu Asp

115 120 125

Val Trp Gly His Gly Thr Ala Val Thr Val Ser Ser

130 135 140

<210> 105

<211> 112

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(112)

<223> VL domain of humanized anti-HIV-1 Env gp120 antibody PGT145

<400> 105

Glu Val Val Ile Thr Gln Ser Pro Leu Phe Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Ala Ala Ser Leu Ser Cys Lys Cys Ser His Ser Leu Gln His Ser

20 25 30

Thr Gly Ala Asn Tyr Leu Ala Trp Tyr Leu Gln Arg Pro Gly Gln Thr

35 40 45

Pro Arg Leu Leu Ile His Leu Ala Thr His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ser Asp Asp Val Gly Thr Tyr Tyr Cys Met Gln Gly

85 90 95

Leu His Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 106

<211> 121

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(121)

<223> VH Domain of human anti-HIV-1 Env gp120 antibody VRC01

<400> 106

Gln Val Gln Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu

1 5 10 15

Ser Met Arg Ile Ser Cys Arg Ala Ser Gly Tyr Glu Phe Ile Asp Cys

20 25 30

Thr Leu Asn Trp Ile Arg Leu Ala Pro Gly Lys Arg Pro Glu Trp Met

35 40 45

Gly Trp Leu Lys Pro Arg Gly Gly Ala Val Asn Tyr Ala Arg Pro Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Val Tyr Ser Asp Thr Ala Phe

65 70 75 80

Leu Glu Leu Arg Ser Leu Thr Val Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Thr Arg Gly Lys Asn Cys Asp Tyr Asn Trp Asp Phe Glu His Trp Gly

100 105 110

Arg Gly Thr Pro Val Ile Val Ser Ser

115 120

<210> 107

<211> 104

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(104)

<223> VL domain of human anti-HIV-1 Env gp120 antibody VRC01

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Thr Ala Ile Ile Ser Cys Arg Thr Ser Gln Tyr Gly Ser Leu Ala

20 25 30

Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Ser

35 40 45

Gly Ser Thr Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg

50 55 60

Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp

65 70 75 80

Phe Gly Val Tyr Tyr Cys Gln Gln Tyr Glu Phe Phe Gly Gln Gly Thr

85 90 95

Lys Val Gln Val Asp Ile Lys Arg

100

<210> 108

<211> 131

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(131)

<223> VH Domain of human anti-HIV-1 gp41 antibody 10E8

<400> 108

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Asp Asn Ala

20 25 30

Trp Met Thr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Thr Gly Pro Gly Glu Gly Trp Ser Val Asp Tyr Ala Ala

50 55 60

Pro Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Asn Ser Ile Asn Phe

65 70 75 80

Leu Tyr Leu Glu Met Asn Asn Leu Arg Met Glu Asp Ser Gly Leu Tyr

85 90 95

Phe Cys Ala Arg Thr Gly Lys Tyr Tyr Asp Phe Trp Ser Gly Tyr Pro

100 105 110

Pro Gly Glu Glu Tyr Phe Gln Asp Trp Gly Arg Gly Thr Leu Val Thr

115 120 125

Val Ser Ser

130

<210> 109

<211> 109

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(109)

<223> VL domain of human anti-HIV-1 gp41 antibody 10E8

<400> 109

Ser Tyr Glu Leu Thr Gln Glu Thr Gly Val Ser Val Ala Leu Gly Arg

1 5 10 15

Thr Val Thr Ile Thr Cys Arg Gly Asp Ser Leu Arg Ser His Tyr Ala

20 25 30

Ser Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Ile Leu Leu Phe Tyr

35 40 45

Gly Lys Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Ala Ser Gly Asn Arg Ala Ser Leu Thr Ile Ser Gly Ala Gln Ala Glu

65 70 75 80

Asp Asp Ala Glu Tyr Tyr Cys Ser Ser Arg Asp Lys Ser Gly Ser Arg

85 90 95

Leu Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 110

<211> 509

<212> PRT

<213> Artificial sequence

<220>

<223> first polypeptide chain of DART-1

<400> 110

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Ile His Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Arg Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Leu Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Asn Leu Gln Ala Glu Asp Val Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly His Gly Thr Arg Val Glu Ile

100 105 110

Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr

165 170 175

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

210 215 220

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Glu Val Ala Ala Cys

245 250 255

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

260 265 270

Glu Val Ala Ala Leu Glu Lys Gly Gly Gly Asp Lys Thr His Thr Cys

275 280 285

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

290 295 300

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

305 310 315 320

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

325 330 335

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

340 345 350

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

355 360 365

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

370 375 380

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

385 390 395 400

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

405 410 415

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

420 425 430

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

435 440 445

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

450 455 460

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

465 470 475 480

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

485 490 495

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505

<210> 111

<211> 277

<212> PRT

<213> Artificial sequence

<220>

<223> second polypeptide chain of DART-1

<400> 111

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly

115 120 125

Val Phe Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly

130 135 140

Phe Thr Phe Thr Glu Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Leu Ala Tyr Ile Ser Lys Asn Gly Glu Tyr Ser

165 170 175

Lys Tyr Ser Pro Ser Ser Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn

180 185 190

Ala Lys Asn Ser Val Phe Leu Gln Leu Asp Arg Leu Ser Ala Asp Asp

195 200 205

Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asp Gly Leu Thr Tyr Phe Ser

210 215 220

Glu Leu Leu Gln Tyr Ile Phe Asp Leu Trp Gly Gln Gly Ala Arg Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Lys Val Ala Ala Cys Lys Glu

245 250 255

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val

260 265 270

Ala Ala Leu Lys Glu

275

<210> 112

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> third polypeptide chain of DART-1

<400> 112

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 113

<211> 509

<212> PRT

<213> Artificial sequence

<220>

<223> first polypeptide chain of DART-A

<400> 113

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Ser Lys Tyr

165 170 175

Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn

210 215 220

Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Glu Val Ala Ala Cys

245 250 255

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

260 265 270

Glu Val Ala Ala Leu Glu Lys Gly Gly Gly Asp Lys Thr His Thr Cys

275 280 285

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

290 295 300

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

305 310 315 320

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

325 330 335

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

340 345 350

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

355 360 365

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

370 375 380

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

385 390 395 400

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

405 410 415

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

420 425 430

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

435 440 445

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

450 455 460

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

465 470 475 480

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

485 490 495

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505

<210> 114

<211> 277

<212> PRT

<213> Artificial sequence

<220>

<223> second polypeptide chain of DART-A

<400> 114

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Glu Tyr Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Lys Asn Gly Glu Tyr Ser

165 170 175

Lys Tyr Ser Pro Ser Ser Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn

180 185 190

Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

195 200 205

Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asp Gly Leu Thr Tyr Phe Ser

210 215 220

Glu Leu Leu Gln Tyr Ile Phe Asp Leu Trp Gly Gln Gly Thr Leu Val

225 230 235 240

Thr Val Ser Ser Ala Ser Thr Lys Gly Lys Val Ala Ala Cys Lys Glu

245 250 255

Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val

260 265 270

Ala Ala Leu Lys Glu

275

<210> 115

<211> 502

<212> PRT

<213> Artificial sequence

<220>

<223> first polypeptide chain of DART-B

<400> 115

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Gly Gly Gly Ser Gly Gly Gly Gly Gln Val Thr Leu Arg Glu Ser

115 120 125

Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr

130 135 140

Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile

145 150 155 160

Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp

165 170 175

Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile

180 185 190

Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met

195 200 205

Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gln Ile Asn Pro Ala

210 215 220

Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

225 230 235 240

Ser Thr Lys Gly Glu Val Ala Ala Cys Glu Lys Glu Val Ala Ala Leu

245 250 255

Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys

260 265 270

Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

275 280 285

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

290 295 300

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

305 310 315 320

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

325 330 335

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

340 345 350

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

355 360 365

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

370 375 380

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

385 390 395 400

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

405 410 415

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

420 425 430

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

435 440 445

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

450 455 460

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

465 470 475 480

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

485 490 495

Ser Leu Ser Pro Gly Lys

500

<210> 116

<211> 278

<212> PRT

<213> Artificial sequence

<220>

<223> second polypeptide chain of DART-B

<400> 116

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Glu Ser Gly Gly

115 120 125

Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

130 135 140

Gly Phe Thr Phe Ser Glu Tyr Tyr Met Thr Trp Ile Arg Gln Ala Pro

145 150 155 160

Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Lys Asn Gly Glu Tyr

165 170 175

Ser Lys Tyr Ser Pro Ser Ser Asn Gly Arg Phe Thr Ile Ser Arg Asp

180 185 190

Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

195 200 205

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Asp Gly Leu Thr Tyr Phe

210 215 220

Ser Glu Leu Leu Gln Tyr Ile Phe Asp Leu Trp Gly Gln Gly Thr Leu

225 230 235 240

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Lys Val Ala Ala Cys Lys

245 250 255

Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys

260 265 270

Val Ala Ala Leu Lys Glu

275

<210> 117

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> third polypeptide chain of TRIDENT-A

<400> 117

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Leu Ile Tyr Tyr Asp Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Pro His Tyr Asp Gly Tyr Tyr His Phe Phe Asp Ser Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 118

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> fourth polypeptide chain of TRIDENT-A

<400> 118

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Gly Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asp Ile Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Gln Tyr Asn Asn Gly Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 119

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> third polypeptide chain of TRIDENT-B

<400> 119

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Ser Cys Thr Val Ser Gly Gly Ser Ser Ser Ser Gly

20 25 30

Ala His Tyr Trp Ser Trp Ile Arg Gln Tyr Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile His Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Ile Thr Ile Ser Gln His Thr Ser Glu Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Val Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Thr Arg Leu Arg Thr Leu Arg Asn Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala

225 230 235 240

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 120

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<223> fourth polypeptide chain of TRIDENT-B

<400> 120

Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Ile Ile Ser Glu Val Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Ser Ser Tyr Thr Asp Ile

85 90 95

His Asn Phe Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 121

<211> 456

<212> PRT

<213> Artificial sequence

<220>

<223> third polypeptide chain of TRIDENT-D

<400> 121

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Glu Tyr

20 25 30

Tyr Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Lys Asn Gly Glu Tyr Ser Lys Tyr Ser Pro Ser Ser

50 55 60

Asn Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Asp Gly Leu Thr Tyr Phe Ser Glu Leu Leu Gln Tyr Ile

100 105 110

Phe Asp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225 230 235 240

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

355 360 365

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val

405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys

435 440 445

Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 122

<211> 220

<212> PRT

<213> Artificial sequence

<220>

<223> fourth polypeptide chain of TRIDENT-D

<400> 122

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser

20 25 30

Ser Asn Asn Arg His Ser Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Met Arg Leu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Ser Ser His Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 123

<211> 101

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(101)

<223> VL domain of framework 1-3 Donor (IGKV4-1)

<400> 123

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro

100

<210> 124

<211> 10

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> VL Domain of framework 4 Donor (IGKJ1-01)

<400> 124

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 125

<211> 98

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(98)

<223> VH Domain of framework 1-3 Donor (IGVH3-11)

<400> 125

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Tyr Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 126

<211> 11

<212> PRT

<213> Intelligent people

<220>

<221> MISC_FEATURE

<222> (1)..(11)

<223> VH Domain of framework 4 Donor (IGHJ4-01)

<400> 126

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 127

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD16 antibody hCD16-M1

<400> 127

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Val Arg Gln Ser Ala Arg Ala Pro Glu Pro Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 128

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD16 antibody hCD16-M1

<400> 128

Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Gly Thr His

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Leu

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 129

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> VH Domain of humanized anti-human CD16 antibody CD16-M2VH 1

<400> 129

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn His Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gly Tyr Arg Tyr Ala Ser Trp Phe Ala Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 130

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD16 antibody CD16-M2VH2

<400> 130

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn His Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Arg Tyr Ala Ser Trp Phe Ala Ser Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 131

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain of humanized anti-human CD16 antibody hCD16-M2 VL1

<400> 131

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Ser Val Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105