阅读说明：本技术 结合分子 (Binding molecules ) 是由 格兰尼·邓利维 科莉特·约翰斯顿 丹妮拉·西多鲁克 马蒂纳·莱万多夫斯卡 于 2020-05-15 设计创作，主要内容包括：本公开内容涉及结合人血清白蛋白的单结构域抗体和使用此类单结构域抗体来延长治疗分子的半寿期的方法。(The present disclosure relates to single domain antibodies that bind human serum albumin and methods of using such single domain antibodies to extend the half-life of therapeutic molecules.)

1. An immunoglobulin single variable domain antibody that binds to human HSA comprising

a) CDR1 having SEQ ID NO 2 or an amino acid sequence with 1 or 2 differences from SEQ ID NO 2;

b) CDR2 having SEQ ID NO. 3 or an amino acid sequence that differs from SEQ ID NO. 3 by 1,2,3, 4,5, 6; and

c) CDR3 having SEQ ID NO. 4 or an amino acid sequence with 1,2,3 or 4 differences from SEQ ID NO. 4.

2. The immunoglobulin single variable domain antibody according to claim 1, comprising or consisting of SEQ ID No.1 or a sequence having at least 80%, 90% or 95% homology to SEQ ID No. 1.

3. The immunoglobulin single variable domain antibody according to claim 2, comprising or consisting of SEQ ID NO 30 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 30.

4. An immunoglobulin single variable domain antibody that binds to human HSA comprising

d) CDR1 having SEQ ID NO 6 or an amino acid sequence with 1 or 2 differences from SEQ ID NO 6;

e) CDR2 having SEQ ID NO. 7 or an amino acid sequence that differs from SEQ ID NO. 7 by 1,2,3, 4,5, 6; and

f) CDR3 having SEQ ID NO 8 or an amino acid sequence with 1,2,3 or 4 differences from SEQ ID NO 8.

5. The immunoglobulin single variable domain antibody according to claim 4, comprising or consisting of SEQ ID NO 5 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 5.

6. A protein or construct comprising an immunoglobulin single variable domain according to the preceding claim.

7. The protein or construct of claim 6, comprising a therapeutic moiety.

8. The protein or construct of claim 7, wherein the therapeutic moiety is an antibody or fragment thereof.

9. The protein or construct of claim 8, wherein the fragment is an scFv, Fv, heavy chain or single domain antibody.

10. The protein or construct of claim 9, wherein the single domain antibody is a single variable heavy domain antibody.

11. The protein or construct of any one of claims 6 to 10, wherein the immunoglobulin single variable domain antibody is linked to the therapeutic moiety by a peptide linker.

12. The protein or construct of claim 10, wherein the peptide linker is (G4S) n, wherein n is 1 to 15.

13. The protein or construct of any of claims 6 to 12, wherein the immunoglobulin single variable domain antibody is located at the N-or C-terminus of the protein.

14. The protein or construct of claim 13, wherein the immunoglobulin single variable domain antibody is located C-terminal to the protein and comprises a C-terminal extension of 1 to 50 amino acids.

15. A method for extending the half-life of a protein comprising linking said protein to an immunoglobulin single variable domain antibody according to any one of claims 1 to 5.

16. Use of an immunoglobulin single variable domain antibody according to any one of claims 1 to 5, for prolonging the half-life of a therapeutic moiety in a fusion protein when the immunoglobulin single variable domain according to any one of claims is linked to said therapeutic moiety.

17. A pharmaceutical composition comprising an immunoglobulin single variable domain according to any one of claims 1 to 5 or a protein or construct according to any one of claims 6 to 14.

18. Nucleic acid sequence encoding the amino acid sequence according to any one of claims 1 to 5.

19. The nucleic acid sequence of claim 18, comprising SEQ ID NO 16.

20. The nucleic acid sequence of claim 18 or 19, wherein the nucleic acid sequence is linked to a second nucleic acid sequence by a linker.

21. The nucleic acid sequence of claim 20, wherein the second nucleic acid encodes a therapeutic moiety.

22. The nucleic acid sequence of claim 20 or 21, wherein the linker is a nucleic acid linker.

23. A vector comprising a nucleic acid sequence according to any one of claims 18 to 22.

24. A host cell comprising a nucleic acid sequence according to any one of claims 18 to 22 or a vector according to claim 23.

25. A kit comprising an immunoglobulin single variable domain antibody according to any one of claims 1 to 5 or a protein or construct according to any one of claims 6 to 14 or a pharmaceutical composition according to claim 16.

26. Method for the production of a binding molecule comprising at least one human immunoglobulin single domain antibody according to any of claims 6 to 14 capable of binding to human HSA, wherein said domain is human V_HA domain, said method comprising

a) Immunizing a transgenic mouse with an HSA antigen, wherein the mouse expresses a nucleic acid construct comprising a human heavy chain V gene and is incapable of producing a functional endogenous light or heavy chain, b) generating a polypeptide comprising a V from the mouse_HA sequence library of domain sequences, and

c) isolation of a peptide comprising V from the library_HThe sequence of the domain sequence.

Summary of The Invention

The present invention relates to immunoglobulin single variable domain antibodies, particularly human immunoglobulin single variable heavy domain antibodies, that bind HSA, e.g., particularly human immunoglobulin single variable heavy domain antibodies obtained or obtainable from transgenic mice expressing unrearranged human V, D, J gene segments.

In one aspect, the invention relates to an immunoglobulin single variable domain antibody that binds HSA, comprising or consisting of: SEQ ID NO 1 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 1, SEQ ID NO 30 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 30, or SEQ ID NO 5 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 5. The invention also relates to an immunoglobulin single variable domain that binds HSA, which is a variant of SEQ ID No.1 and has 1 to 20 amino acid substitutions compared to SEQ ID No. 1. The invention also relates to an immunoglobulin single variable domain that binds HSA, which is a variant of SEQ ID No.5 and has 1 to 20 amino acid substitutions compared to SEQ ID No. 5.

In another aspect, the invention also relates to a method for extending the half-life of a protein comprising linking said protein to an immunoglobulin single variable domain as described herein.

The invention also relates to the use of an immunoglobulin single variable domain antibody as described herein to extend the half-life of a therapeutic moiety when said immunoglobulin single variable domain antibody is linked to said therapeutic moiety in a fusion protein.

In another aspect, the invention relates to a pharmaceutical composition comprising an immunoglobulin single variable domain antibody as described herein or a protein or construct as described herein.

The invention also relates to nucleic acid sequences encoding amino acid sequences as described herein.

The invention further relates to a vector comprising a nucleic acid sequence as described herein.

The invention also relates to a host cell comprising a nucleic acid sequence as described herein or a vector as described herein.

The invention also relates to a kit comprising an immunoglobulin single variable domain antibody as described herein or a protein or construct as described herein or a pharmaceutical composition as described herein.

Furthermore, the present invention relates to a method for the production of an antibody with a heavy chain only or a binding molecule comprising at least one human immunoglobulin single domain antibody capable of binding to human HSA as described herein, wherein said domain is human V_HA domain, said method comprising

a) Immunizing a transgenic mouse with an HSA antigen, wherein the mouse expresses a nucleic acid construct comprising a human heavy chain V gene and is incapable of producing a functional endogenous light or heavy chain, b) generating a polypeptide comprising a V from the mouse_HA sequence library of domain sequences, and

c) isolation of a peptide comprising V from the library_HThe sequence of the domain sequence.

Drawings

The invention is further illustrated in the following non-limiting figures.

FIG. 1: serum levels after a single intravenous administration of TPP-1246 at 3mg/kg in cynomolgus monkeys.

Detailed Description

Embodiments of the present invention will now be further described. In the following paragraphs, different embodiments are described. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

Generally, the terms and techniques used in connection with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, fourth edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); therapeutic Monoclonal Antibodies From Bench to clinical, Zhijiang An (main edition), Wiley (2009); and Antibody Engineering, second edition, volumes 1 and 2, compiled by Ontermann and Dubel, Springer-Verlag, Heidelberg (2010).

Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature used, and the laboratory procedures and techniques, in connection with the analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

The present invention relates to amino acid sequences that bind to Human Serum Albumin (HSA) and binding molecules, such as proteins, comprising such amino acid sequences. In particular, the present invention relates to single domain antibodies or immunoglobulin single variable domains having amino acids as described herein and useful in therapeutic methods and uses and pharmaceutical formulations as described herein.

The single domain antibodies described herein specifically bind to wild-type human serum albumin (UniProt accession No. Q56G 89). The amino acid sequence of wild-type human serum albumin is shown in SEQ ID NO 9.

Human serum albumin (HSA, 2BXN) contains about 60% of plasma proteins. HSA consists of a single chain, 585 amino acids in length, and contains three homology domains (I, II and III). Domain I consists of residues 5-197, domain II comprises residues 198-382 and domain III is formed by residues 383-569. Each domain comprises two subdomains designated A and B (IA; residues 5-107, IIA; residues 108-197, IIA; residues 198-296, IIB; residues 297-382, IIIA; residues 383-494, IIIB; residues 495-569).

A single domain antibody (sdAb), immunoglobulin single variable domain or protein of the invention that "binds" or "is capable of binding" an antigen of interest (e.g., human serum albumin) is an antibody that binds the antigen with sufficient affinity such that the single domain antibody can be used as a therapeutic agent to target cells or tissues expressing the antigenic human serum albumin as described herein.

The single domain antibodies, immunoglobulin single variable domains, or proteins described herein specifically bind to human serum albumin. In other words, binding to human serum albumin antigen is clearly distinct from non-specific interactions. As shown in the examples, single domain antibodies do not cross-react with mouse human serum albumin. Preferably, the single domain antibody binds to human serum albumin and also binds to monkey serum albumin as shown in the examples.

As used herein, the term "antibody" broadly refers to any immunoglobulin (Ig) molecule or antigen-binding portion thereof comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding characteristics of an Ig molecule.

In a full-length antibody, each heavy chain comprises a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region comprises three domains C_H1、C_H2 and C_H3. Each light chain comprises a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region comprises a domain C_L。

The heavy and light chain variable regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FRs). Each heavy and light chain variable region contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.

The immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass. The term "CDR" refers to complementarity determining regions within an antibody variable sequence. There are three CDRs in each variable region of the heavy and light chains, called CDR1, CDR2, and CDR3, respectively, for each variable region. The term "set of CDRs" refers to a set of three CDRs in a single variable region that is now capable of binding antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

Kabat Complementarity Determining Regions (CDRs) are most commonly used based on sequence variability (Kabat et al, (1971) Ann. NY Acad. Sci.190: 382-plus 391 and Kabat et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers to the position of the structural loops (Chothia and Lesk J.mol.biol.196:901-917 (1987)). When referring to residues in the variable domain (approximately residues 1-107 for the light chain and residues 1-113 for the heavy chain), the Kabat numbering system is typically used. Another system is the ImmunoGeneTiCs (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al, Dev. Comp. Immunol.,29,185-203 (2005).

The system described by Kabat is used herein. The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms are art-recognized and refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding portion.

The term "antigen binding site" refers to a portion of an antibody or antibody fragment that comprises a region that specifically binds to an antigen. The antigen binding site may be provided by one or more antibody variable domains. The antigen binding site is typically comprised in the relevant V of the antibody or antibody fragment_HAnd V_LAnd (4) the following steps.

Antibody fragments are part of antibodies, e.g., F (ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (V)_H) Variable light (V)_L) A chain domain, etc. Functional fragments of full-length antibodies retain the target specificity of the intact antibody. Thus, recombinant functional antibody fragments such as Fab (fragments, antibodies), scFv (single chain variable fragments) and single domain antibodies (dabs) have been used to develop therapeutics as alternatives to mAb-based therapeutics.

scFv fragment (. about.25 kDa) consisting of two variable domains V_HAnd V_LAnd (4) forming. Naturally, V_HAnd V_LThe domains are non-covalently associated by hydrophobic interactions and tend to dissociate. However, by linking these domains with hydrophilic flexible linkers to produce single chain fv (scFv), stable fragments can be generatedAnd (5) engineering.

The smallest antigen-binding fragment is the single variable fragment, the variable heavy chain (V)_H) Or variable light chain (V)_L) A domain. V_HAnd V_LThe domains are each capable of binding an antigen. Target binding does not require binding to the light/heavy chain partners, respectively, or the presence of other portions of the virtually intact antibody. Size reduction to a single domain (corresponding to V)_HOr V_LDomains) are commonly referred to as "single domain antibodies" or "immunoglobulin single variable domains". Thus, single domain antibodies (. about.12 to 15kDa) have a V_HOr V_LA domain, i.e., it has no other portion of an intact antibody. Single domain antibodies derived from antibodies with camelid heavy chains only naturally lacking light chains have been described as well as single domain antibodies with human heavy chain domains. Murine V that has also been amplified, e.g., from genomic DNA of the spleen of an immunized mouse_HIdentification of antigen-binding Single V in Gene libraries_HDomain, and expressed in E.coli (Ward et al, 1989, Nature 341: 544-546). Ward et al name isolated Single V_HThe domain "dAb" is a "domain antibody". The term "dAb" generally refers to a single immunoglobulin variable domain (V) that specifically binds an antigen_H、V_HHOr V_L) A polypeptide. For use in therapy, human single domain antibodies are compared to camelidae-derived V_HHMore preferably, primarily because they are less likely to elicit an immune response when administered to a patient.

The term "single domain antibody, single variable domain, or immunoglobulin single variable domain (ISV)" is well known in the art and describes a single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. "Single heavy chain Domain antibody, Single variable heavy chain Domain, immunoglobulin Single heavy chain variable Domain (ISV), human V_HSingle domain "describes a single heavy chain variable fragment of an antibody that retains binding specificity for an antigen in the absence of a light chain or other antibody fragment. A single variable heavy domain antibody does not comprise any other chain of a full length antibody; it has no any lightnessA chain or a constant domain. Thus, it is capable of binding antigen in the absence of a light chain.

In one aspect, the invention relates to immunoglobulin single variable domains that bind human serum albumin. As described below, embodiments relate to single variable heavy domain antibodies/immunoglobulin single variable heavy domains that bind to HSA antigen. Thus, a single variable heavy domain antibody is capable of binding to HSA in the absence of a light chain. Human single variable heavy domain antibodies (' V)_HSingle domain antibody/single V_HDomain antibodies ") are particularly preferred. Such binding molecules are also referred to herein asIs a registered trademark of crescando Biologics ltd.

Thus, in some embodiments, an isolated binding agent/molecule comprises or consists of at least one single domain antibody, wherein said domain is a human immunoglobulin variable heavy chain domain; they have no V_LA domain or other antibody fragment, and binds to a target antigen.

The term "isolated" refers to a moiety that is separated from its natural environment. For example, the term "isolated" refers to a single domain antibody that is substantially free of other single domain antibodies, or antibody fragments. Furthermore, the isolated single domain antibody may be substantially free of other cellular material and/or chemicals.

Each V_HThe domain antibody comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (V) having the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4_H) A domain.

Single domain antibodies of the invention may be subjected to C-or N-terminal V_HModification of the framework sequence to improve its properties. For example, V_HThe domain may comprise a C or N terminal extension. Capable of adding a C-terminal extension to a V terminated with residues VTVSS (SEQ ID NO:10)_HThe C-terminus of the domain.

In one embodiment, a single domain antibody of the invention comprises a C-terminal extension of 1 to 50 residues, such as 1 to 10, e.g., 1,2,3, 4,5,6,7,8,9 or 10, 1-20, 1-30 or 1-40 additional amino acids. In one embodiment, the single domain antibody of the invention comprises human C_H1 domain, such that the C-terminus extends to C_H1 domain. For example, a C-terminal extension may comprise a neutral, non-polar amino acid, such as A, L, V, P, M, G, I, F or W, or a neutral polar amino acid, such as S or T. The C-terminal extension may also be selected from a peptide linker or tag. In addition, the C or N terminal residue may be, for example, a peptide linker for conjugating the single domain antibody of the invention to another moiety, or a tag to facilitate detection of the molecule. Such tags are well known in the art and include, for example, a linker His tag, such as a hexa-His (HHHHHHHH, SEQ ID NO:11) or myc tag.

As used herein, the term "homology" or "identity" generally refers to the percentage of amino acid residues in a sequence that are identical to the residues of a reference polypeptide to which it is compared, after aligning the sequences, in some embodiments, after introducing gaps (if necessary) to achieve the maximum percent homology, and without considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N-or C-terminal extension, tag, or insertion should be construed as reducing identity or homology. Methods and computer programs for alignment are well known. The percent identity between two amino acid sequences can be determined using well-known mathematical algorithms.

The variable domain of a single domain antibody as described herein is fully human or substantially fully human. As used herein, the term V_HDomain antibodies represent single human variable heavy chain domain antibodies (and V representing the Camelidae heavy chain domain)_HHThe opposite). As used herein, human V_HThe domain comprises a fully human or substantially fully human V_HA domain. As used hereinBy the term human V_HDomains also include V isolated from heavy chain-only antibodies_HDomains prepared from transgenic mice expressing fully human immunoglobulin heavy chain loci, particularly in response to immunization with an antigen of interest (i.e., HSA), e.g., as described in WO2016/062990 and the examples below. In one embodiment, human V_HDomains can also include derived from or based on human V_HDomain amino acids or from human V_HV produced by nucleic acid sequence_HA domain. Thus, the term human V_HThe domains include variable heavy chain regions derived from or encoded by human immunoglobulin sequences, and are obtained, for example, from heavy chain-only antibodies produced in transgenic mice expressing fully human unrearranged V, D, J gene segments. In some embodiments, substantially human V_HDomains are either derived from or based on human V_HV of the Domain_HThe domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, such as by random or site-specific mutagenesis, or by somatic mutation in vivo). Thus the term "human V_HDomain "also includes substantially human V_HA domain in which one or more amino acid residues have been modified, for example to remove sequence susceptibility. For example, substantially human V as compared to germline human sequences_HA domain may comprise up to 10, for example 1,2,3, 4,5,6,7,8,9 or 10 or up to 20 amino acid modifications.

However, as used herein, the term "human V_HDomain "or" substantially human V_HThe "domain" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In one embodiment, as used herein, the term "human V_HDomains "are also not intended to include camelized V_HDomains, i.e.having been selected for V, e.g.in vitro, by conventional mutagenesis methods_HAt a predetermined position in the domain sequence and introducing one or more point mutations at the predetermined position to alter one or more predetermined residuesBecome available in camelidae V_HHHuman V specifically modified at specific residues found in the Domain_HA domain.

The molecules of the invention are advantageous because they are fully human and therefore non-immunogenic. They do not require humanization (humanization).

In a first aspect, there is provided an immunoglobulin single variable domain antibody comprising

a) CDR1 having SEQ ID NO 2 or an amino acid sequence that differs from SEQ ID NO 2 by 1,2,3, 4,5 or 5

b) CDR2 having SEQ ID NO 3 or an amino acid sequence which differs from SEQ ID NO 3 by 1,2,3, 4,5, 7,8,9,10,11,12, 13, 14, 15, 16 or 17, and/or

c) CDR3 having SEQ ID NO. 4 or an amino acid sequence that differs from SEQ ID NO. 4 by 1,2,3, 4,5,6,7,8,9,10,11,12, 13 or 14.

In one embodiment, the immunoglobulin single variable domain has one of the CDRs defined above, e.g., CDR1, CDR2 or CDR 3. In one embodiment, the CDRs are selected from SEQ ID NOs 2,3, or 4, respectively. In another embodiment, the CDRs are variants and have substitutions as defined above. In another embodiment, one or both CDR sequences are as defined from SEQ ID NOs 2,3 or 4 and the remaining CDRs are the respective CDR sequences 2,3 or applicable variants.

In one embodiment, the single variable domain antibody comprises or consists of: 1 or a sequence having at least 80%, 90% or 95% homology to SEQ ID No. 1.

SEQ ID NO 1 is shown below:

(SEQ ID NO:1, also referred to herein as)

The sequences of CDR1, CDR2, and CDR3 are shown in bold above, respectively. The CDRs have the following sequences:

NYNMN CDR1:(SEQ ID NO:2)

SISSAGTHIYSADSVKG CDR2:(SEQ ID NO:3)

DPHSTGWYKDFDY CDR3:(SEQ ID NO:4)

in one embodiment, there is provided a single variable domain antibody capable of binding to human serum albumin and having 4 framework regions (FR 1 to FR4, respectively) and 3 complementarity determining regions (CDR 1 to CDR3, respectively), wherein:

(i) CDR1 comprises or is the amino acid sequence set forth in SEQ ID NO. 2; CDR2 comprises or is the amino acid sequence SEQ ID NO 3; and CDR3 comprises or is the amino acid sequence SEQ ID NO 4, and wherein

(ii) The amino acid sequence has at least 85%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO. 1.

In another aspect, immunoglobulin single variable domain antibodies are provided, comprising

a) CDR1 having SEQ ID NO 6 or an amino acid sequence that differs from SEQ ID NO 6 by 1,2,3, 4,5 or 5

b) CDR2 having SEQ ID NO 7 or an amino acid sequence that differs from SEQ ID NO 7 by 1,2,3, 4,5, 7,8,9,10,11,12, 13, 14, 15, 16 or 17, and/or

c) CDR3 having SEQ ID NO 8 or an amino acid sequence that differs from SEQ ID NO 8 by 1,2,3, 4,5,6,7,8,9,10,11,12, 13 or 14.

In one embodiment, the immunoglobulin single variable domain has one of the CDRs defined above, e.g., CDR1, CDR2 or CDR 3. In one embodiment, the CDRs are selected from SEQ ID NO 6,7 or 8, respectively. In another embodiment, the CDRs are variants and have substitutions as defined above. In another embodiment, one or both CDR sequences are as defined from SEQ ID NOs 6,7 or 8 and the remaining CDRs are the respective CDR sequences 6,7,8 or variants as applicable.

In one embodiment, the single variable domain antibody comprises or consists of: SEQ ID NO 5 or a sequence having at least 80%, 90% or 95% homology to SEQ ID NO 5.

SEQ ID NO 5 is shown below:

(SEQ ID NO:5, also referred to herein as)

The sequences of CDR1, CDR2, and CDR3 are shown in bold above, respectively. The CDRs have the following sequences:

SYTMN CDR1:(SEQ ID NO:6)

SISSSGRYIYYADSVKG CDR2:(SEQ ID NO:7)

DPRMVGNPHEFDI CDR3:(SEQ ID NO:8)

in one embodiment, there is provided a single variable domain antibody capable of binding to human serum albumin and having 4 framework regions (FR 1 to FR4, respectively) and 3 complementarity determining regions (CDR 1 to CDR3, respectively), wherein:

(i) CDR1 comprises or is the amino acid sequence set forth in SEQ ID NO. 6; CDR2 comprises or is the amino acid sequence SEQ ID NO. 7; and CDR3 comprises or is the amino acid sequence SEQ ID NO 8, and wherein

(ii) The amino acid sequence has at least 85%, 90% or 95% sequence identity with the amino acid sequence of SEQ ID NO. 5.

As used above, sequence homology/identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, for example at least 95%, 96%, 97%, 98% or 99% sequence homology/identity.

The immunoglobulin single variable domain antibody may be a variant of SEQ ID NO.1 or SEQ ID NO.5 having one or more amino acid substitutions, deletions, insertions or other modifications, and retaining the biological function of the single domain antibody, i.e.HSA binding. Thus, variant V_HSingle domain antibodies can be sequence engineered. Modifications may include one or more substitutions, deletions or insertions of one or more codons encoding a single domain antibody or polypeptide, with native sequence V_HSingle domain antibodies or polypeptides result in changes in the amino acid sequence compared to the polypeptide. An amino acid substitution may be the result of substituting one amino acid with another having similar structural and/or chemical properties, such as substituting leucine with serine, i.e., a conservative amino acid substitution. Insertions or deletions may optionally be in the range of about 1 to 25 amino acids, e.g., 1 to 5, 1 to 10,1 to 15, 1 to 20 amino acids, e.g., 1,2,3, 4,5,6,7,8,9, or 10 amino acids. The variation allowed can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the full-length or mature native sequence. V as described herein_HVariants of a single domain antibody have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology/identity to a non-variant molecule. In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modification" means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the sdabs of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include the following amino acids: having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chainsChains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CDR region of a single domain antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., HSA binding) using the functional assays described herein.

Thus, these amino acid changes can generally be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or specificity for an antigen. Typically, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity. Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to destroy the biological activity of the polypeptide. Abbreviations that contain the amino acid residues of the polypeptides and peptides described herein, as well as conservative substitutions of these amino acid residues, are shown in table 1 below.

TABLE 1 examples of amino acid residues and conservative amino acid substitutions

In some embodiments, the invention provides V_HSingle domain antibody which is V compared to SEQ ID NO 1, SEQ ID NO 30 or SEQ ID NO 5_HA variant of a single domain antibody comprising one or more sequence modifications and having an improvement in one or more properties (e.g., binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility) as compared to an unmodified single domain antibody.

The skilled artisan will appreciate that there are different ways to identify, obtain and optimize antigen binding molecules as described herein, including in vitro and in vivo expression libraries. This is further described in the examples. Optimization techniques known in the art can be used, such as display (e.g., ribosome and/or phage display) and/or mutagenesis (e.g., error-prone mutagenesis). The invention therefore also encompasses sequence-optimized variants of the single domain antibodies described herein.

In one embodiment, modifications can be made to reduce the immunogenicity of the single domain antibody. For example, one approach is to restore one or more framework residues to the corresponding human germline sequence. More specifically, a single domain antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the single domain antibody is derived. These residues can be identified by comparing single domain antibody framework sequences to the germline sequence from which the single domain antibody was derived. In one embodiment, all framework sequences are germline sequences.

In order to return one or more amino acid residues in the framework region sequence to its germline configuration, somatic mutations can be "back-mutated" to a germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

Another type of framework modification involves mutating one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody.

In another embodiment, glycosylation is modified. For example, non-glycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This glycosylation can increase the affinity of the antibody for the antigen.

In one embodiment, one or more substitutions are in the CDR1, 2 or 3 region. For example, there may be 1,2,3, 4,5 or more amino acid substitutions in CDR1, 2 or 3. In another example, there may be 1 or 2 amino acid deletions. In one embodiment, one or more substitutions are in a framework region. For example, there may be 1 to 10 or more amino acid substitutions in the framework regions.

In one embodiment, the variant comprises a substitution at one or more of the following positions or one or more of the following substitutions or a combination thereof with respect to SEQ ID No. 1: e1, V5, G44, Y60,92A,28T → A N31, N33, a54, G55, H57, I58 and/or Y106. Positions are according to Kabat.

In one embodiment, the variant comprises a substitution in respect of SEQ ID No.1 at one or more of the following positions or one or more of the following substitutions or a combination thereof, for example at the following positions: e1 → Q; 5V-L; g44 → R; y60 → S; 92A → G; 28T → A; n31 → S; n33 → T; a54 → G; g55 → S; h57 → Y; i58 → K and/or Y106 → F. Positions are according to Kabat.

In one embodiment, a variant comprises 1,2,3,3,4,5,6,7,8,9,10,11,12 or 13 of the modifications listed above. Combinations of modifications are therefore specifically contemplated.

In one embodiment, the variant comprises one or more of the following substitutions for SEQ ID No.5, or combinations thereof, for example at the following positions: v5, T28, N84, S50, S54, G55, R56, Y60, L103, E108 and/or I111. Positions are according to Kabat.

A variant of SEQ ID NO 1 according to the invention is shown below in SEQ ID NO 30.

The corresponding nucleic acids are shown below (SEQ ID NO: 31).

In one embodiment, the variant comprises one or more of the following substitutions for SEQ ID No.5, or combinations thereof, for example at the following positions: v5 → L; t28 → N or A; n84 → H; s50 → A; s54 → N; g55 → S; r56 → T; y60 → H; l103 → V; e108 → A and/or I111 → V. Positions are according to Kabat.

As described, the amino acid sequences provided by the present invention are capable of binding to a protein, in particular capable of specifically (as described herein) binding to human serum albumin. Thus, they can be used as binding units or binding domains for binding to human serum albumin, e.g. in order to confer an increase in the therapeutic compound, moiety or entity half-life (as defined herein).

The term "half-life" as used may generally refer to the time it takes to reduce the serum concentration of an amino acid sequence, compound or polypeptide by 50% in vivo, for example, due to degradation of the sequence or compound by natural mechanisms and/or clearance or sequestration of the sequence or compound. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention may be determined in any manner known per se, e.g. by pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art. The half-life can be expressed using parameters such as t1/2- α, t1/2- β and area under the curve (AUC). The half-lives (t α and t β) and AUC can be determined from the curve of the serum concentration of the conjugate or fusion over time. Thus, the term "half-life" as used herein refers specifically to tl/2- β or to the terminal half-life (where tl/2- α and/or AUC or both may not be considered).

For example, in the first phase (alpha phase), the drug composition (e.g., drug conjugate, non-covalent drug conjugate, drug fusion) is distributed primarily in the patient with some elimination. The second phase (beta phase) is the terminal phase when the pharmaceutical composition (e.g., drug conjugate, non-covalent drug, drug fusion) has been distributed and the serum concentration decreases as the pharmaceutical composition is cleared from the patient. The ta half-life is the half-life of the first phase and the T β half-life is the half-life of the second phase.

The HSA-binding immunoglobulin variable domain of the invention:

can have a serum half-life (denoted t1/2) in humans of 1 to 72 hours, such as 10 hours or more, for example up to 20 hours or 12, 24, 36 or 48 hours, and/or

-when linked to a therapeutic moiety, conferring a human serum half-life of 1-72 hours, such as 10 hours or more, for example up to 20 hours or 12, 24, 36 or 48 hours, to the resulting protein.

In one embodiment, the immunoglobulin variable domain that binds to HSA is inThe half-life of the molecule was extended by about 20 to 78 hours in the HSA/FcRn mouse model as shown in the examples.

In one embodiment, the immunoglobulin variable domain that binds HSA confers a half-life of the molecule in cynomolgus monkeys of about 84 hours. This can be as shown in the examples, for example for the HSA conjugate (binder) of SEQ ID NO: 30.

As shown in example 9, the immunoglobulin variable domain of the present invention that binds to HSA also has excellent storage stability. They are particularly suitable for elongating V_HHalf-life of single domain antibodies.

The invention also relates to binding molecules comprising or consisting of the immunoglobulin single variable domain antibodies described herein, e.g. comprising or consisting of SEQ ID NO 1, SEQ ID NO 30 or SEQ ID NO 5 or and the second part. In one embodiment, the moiety is a therapeutic moiety. The binding molecule may be a polypeptide, protein or construct. Fusion proteins, multivalent and multispecific proteins or constructs comprising the single variable domain antibodies described herein are also provided. The immunoglobulin single variable domain antibodies described herein (e.g., comprising or consisting of SEQ ID NO:1, SEQ ID NO:30 or SEQ ID NO:5) are for use with a moiety that binds another target, e.g., a therapeutic target.

In one embodiment, the therapeutic moiety is a binding molecule, e.g. selected from an antibody or antibody fragment (e.g. Fab, F (ab')2, Fv, single chain Fv fragment (scFv) or single domain antibody, e.g. V_HOr V_HHDomains) or antibody mimetic proteins. In one embodiment, a single domain antibody of the invention may be linked to an antibody Fc region or fragment thereof comprising C_H2 and C_H3 one or both of the domains, and optionally a hinge region. In one embodiment, the method comprisesLess the second part being V_HA domain.

In one embodiment, the protein or polypeptide comprising an immunoglobulin single variable domain that binds HSA and a second portion as described herein is a fusion protein. In one embodiment, the protein or polypeptide comprising an immunoglobulin single variable domain that binds HSA as described herein and a second portion is a drug conjugate.

As used herein, "conjugate" refers to a composition comprising an antigen-binding fragment of an antibody, wherein the antibody binds to serum albumin bound to a drug.

Such conjugates include "drug conjugates" comprising an antigen-binding fragment of an antibody that binds to serum albumin to which the drug is covalently bound, and "non-covalent drug conjugates" comprising an antigen-binding fragment of an antibody that binds to serum albumin to which the drug is non-covalently bound.

As used herein, "drug conjugate" refers to a composition comprising an antigen-binding fragment of an antibody that binds to serum albumin to which a drug is covalently bonded. The drug can be covalently bound to the antigen-binding fragment directly or indirectly through a suitable linker or spacer moiety. The drug can be conjugated to the antigen-binding fragment at any suitable position, e.g., amino-terminal, carboxy-terminal, or through a suitable amino acid side chain.

In one embodiment, the immunoglobulin single variable domain is linked to the second part by a peptide linker or other suitable linker to link the two parts.

The term "peptide linker" refers to a peptide comprising one or more amino acids. The peptide linker comprises 1 to 50 amino acids, for example 1 to 20 amino acids. Peptide linkers are known in the art and non-limiting examples are described herein. Suitable non-immunogenic linker peptides are, for example, linkers comprising G and/or S residues, (G4S) n, (SG4) n or G4(SG4) n peptide linkers, wherein "n" is typically a number between 1 and 10, for example 1,2,3, 4,5,6,7,8,9 or 10. In one embodiment, the peptide is, for example, selected from the group consisting of GGGGS (SEQ ID NO:12), GGGGSGGGGS (SEQ ID NO:13), SGGGGSGGGG (SEQ ID NO:14), GGGGSGGGGSGGGG (SEQ ID NO:15), GSGSGSGSGS (SEQ ID NO:16), GGSGSGSG (SEQ ID NO:17), GGSGSG (SEQ ID NO:18) and GGSG (SEQ ID NO: 19).

The binding agent may be multispecific, e.g., bispecific. In one embodiment, the binding molecule comprises a first V that binds to HSA as described herein_HSingle domain antibodies (V)_H(A) And a second V that binds to another antigen_HSingle domain antibodies (V)_H(B) And thus has the formula: v_H(A)-L-V_H(B)。V_H(A) And V_H(B) Conjugation, i.e. to V, e.g. with a peptide linker_H(B) And (4) connecting. L represents a linker.

Each V_HComprising CDR and FR regions. Thus, the binding molecule may have the formula: FR1(A) -CDR1(A) -FR2(A) -CDR2(A) -FR3(A) -CDR3(A) -FR4(A) -L-FR1(B) -CDR1(B) -FR2(B) -CDR2(B) -FR3(B) -CDR3(B) -FR4 (B).

Single V_HThe order of domains a and B is not particularly limited, such that within the polypeptide of the invention, single variable domain a may be located N-terminally and single variable domain B may be located C-terminally, or vice versa.

In one embodiment, the binding molecule is bispecific. Thus, in one aspect, the present invention relates to a bispecific molecule comprising a single domain antibody as described herein linked to a second functional moiety having a different binding specificity than said single domain antibody.

In one embodiment, the binding molecule, e.g. protein or construct, is multispecific and comprises the additional, i.e. third, fourth, fifth, etc., moieties.

In one embodiment of the multispecific protein, the V that binds to HSA_HThe domain is located at the C-terminus of the protein. In one embodiment of the multispecific protein, the V that binds to HSA_HThe domain is located at the N-terminus of the protein. In one embodiment of the multispecific protein, the V that binds to HSA_HDomains are not located at the termini.

The second or further therapeutic moiety may be selected from moieties that bind, for example, a tumor antigen or an immunooncology target, but the skilled person will appreciate that the invention is not so limited.

The invention also relates to the use of an immunoglobulin single variable domain as described herein to extend the half-life of a therapeutic moiety when said immunoglobulin single variable domain is linked to said therapeutic moiety in a fusion protein according to any one of the claims.

The invention also relates to the use of an immunoglobulin single variable domain as described herein to extend the half-life of a therapeutic moiety when said immunoglobulin single variable domain is linked to said therapeutic moiety in a fusion protein according to any one of the claims. For example, it can be used to extend the half-life of a protein comprising an sdAb that binds CD137 and an sdAb that binds PSMA or PD-1, e.g., as described herein.

An immunoglobulin single variable domain, e.g., a molecule, as described herein can be V_H(A)-V_H(B)-V_H(C),V_H(B)-V_H(A)-V_H(C),V_H(C)-V_H(A)-V_H(B) Or V_H(C)-V_H(B)-V_H(A) Wherein A is an sdAb that binds CD137, B is an sdAb that binds PSMA or PD-1, and C is an immunoglobulin single variable domain as described herein (e.g., SEQ ID NO:1, SEQ ID NO:30, or SEQ ID NO: 5). V_HIs flexible, as explained elsewhere, suitable linkers connect V_HA molecule. Exemplary molecules comprise or consist of a sequence selected from SEQ ID nos. 22, 23, 26, 28, 33, 34 or 35.

In one embodiment, the single variable heavy chain domain antibody is obtained or obtainable from a transgenic rodent expressing a transgene comprising unrearranged human V, D and a J region, in particular a rodent that produces an antibody with only a human heavy chain. In one embodiment, the rodent does not produce functional endogenous light and heavy chains.

Generally, unless otherwise indicated herein, the immunoglobulin single variable domains, polypeptides, proteins and other compounds and constructs referred to herein are intended for use in the prevention or treatment of human (and/or optionally also in warm-blooded animals, especially mammals) diseases or conditions. Thus, in general, the immunoglobulin single variable domains, polypeptides, proteins and other compounds and constructs described herein are preferably such that they can be used as (bio) drugs or other pharmaceutically or therapeutically active compounds and/or pharmaceutical products or compositions, and/or can suitably be part thereof.

Thus, the present invention also relates to a pharmaceutical composition or formulation comprising an immunoglobulin single variable domain polypeptide, protein or construct as described herein, e.g. a binding molecule or fusion protein comprising an HSA binding single domain as described herein. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier. The immunoglobulin single variable domain polypeptide, protein or construct or pharmaceutical composition may be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal (transmucosal), by inhalation or topical administration, especially otic, nasal, ocular or dermal or by inhalation.

Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the composition is administered parenterally.

The pharmaceutically acceptable carrier or vehicle (vehicle) may be particulate such that the composition is in the form of, for example, a tablet or powder. The term "carrier" refers to a diluent, adjuvant, or excipient administered with the drug antibody conjugate of the present invention. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carrier may be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the single domain antibody or composition of the invention and the pharmaceutically acceptable carrier are sterile when administered to an animal. Water is a preferred carrier when the drug antibody conjugates of the invention are administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The compositions of the invention may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired.

The compositions of the invention can be in liquid form, such as solutions, emulsions or suspensions. The liquid can be used for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. When intended for oral administration, the compositions are preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein to be solid or liquid.

As a solid composition for oral administration, the composition may be formulated into the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafer (wafer), and the like. Such solid compositions typically comprise one or more inert diluents. Further, there may be one or more of the following: binders, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose or gelatin; excipients, such as starch, lactose or dextrin, disintegrating agents, such as alginic acid, sodium alginate, corn starch, and the like; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring; and a colorant. When the composition is in the form of a capsule (e.g., a gelatin capsule), it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or delivered by injection. When intended for oral administration, the composition may comprise one or more of sweeteners, preservatives, dyes/colorants and taste enhancers. In the composition for injection administration, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may also be included.

The compositions can take the form of one or more dosage units. In particular embodiments, it is desirable to administer the composition topically to the area in need of treatment, or by intravenous injection or infusion.

The invention further extends to a method for the treatment of a disease, such as cancer, comprising administering a pharmaceutical composition or formulation as described herein or a binding molecule or fusion protein comprising a single domain that binds HSA as described herein. Also envisaged are pharmaceutical compositions or formulations as described herein or binding molecules or fusion proteins comprising a single domain that binds HSA as described herein for use in the treatment of a disease; for example for the treatment of cancer. Also contemplated is the use of a pharmaceutical composition or formulation as described herein or a binding molecule or fusion protein comprising a single domain that binds HSA as described herein in the manufacture of a medicament for the treatment of cancer.

The amount of therapeutic agent that is effective/active in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help determine optimal dosage ranges. The precise dosage employed in the composition will also depend on the route of administration and the severity of the disease or condition, and will be determined at the discretion of the attendant physician and in the individual patient's circumstances. Factors such as age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combination, sensitivity of response, and severity of the disease should be considered.

Typically, the amount is at least about 0.01% of a single domain antibody of the invention by weight of the composition. When intended for oral administration, the amount may vary from about 0.1% to about 80% by weight of the composition. Preferred oral compositions may comprise from about 4% to about 50% of a single domain antibody of the invention by weight of the composition.

Preferred compositions of the invention are prepared such that the parenteral dosage unit contains from about 0.01% to about 2% by weight of a single domain antibody of the invention.

For injectable administration, the compositions can contain from about generally about 0.1mg/kg to about 250mg/kg of subject body weight, preferably from about 0.1mg/kg to about 20mg/kg of animal body weight, and more preferably from about 1mg/kg to about 10mg/kg of animal body weight. In one embodiment, the composition is administered at a dose of about 1 to 30mg/kg, such as about 5 to 25mg/kg, about 10 to 20mg/kg, about 1 to 5mg/kg or about 3 mg/kg. The dosing schedule may vary from, for example, once per week to once every 2,3, or 4 weeks.

As used herein, "treating" or "treatment" refers to inhibiting or ameliorating a disease or disorder. For example, treatment may include delaying the development of symptoms associated with a disease or disorder, and/or reducing the severity of such symptoms that would or are expected to progress with the disease. These terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, the term means that a beneficial result is delivered to at least some of the treated mammals, e.g., human patients. Many drug treatments are effective in some, but not all, patients receiving treatment.

The term "subject" or "patient" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

The molecules or pharmaceutical compositions of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agents. Therapeutic agents are compounds or molecules that can be used to treat diseases. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents (pro-apoptotic agents), anti-angiogenic agents, boron compounds, photosensitizers or dyes, and radioisotopes.

The invention also relates to a method for extending the half-life of a protein comprising linking said protein to an immunoglobulin single variable domain as described herein.

The invention also relates to nucleic acid sequences encoding the amino acid sequences described herein. In one embodiment, the nucleic acid is SEQ ID NO. 20 or a nucleic acid having at least 90% sequence homology to SEQ ID NO. 20. In one embodiment, the nucleic acid is SEQ ID NO 21 or a nucleic acid having at least 90% sequence homology to SEQ ID NO 21. In one embodiment, the nucleic acid sequence is linked to the second nucleic acid sequence by a linker. In one embodiment, the second nucleic acid encodes a therapeutic moiety. In one embodiment, the linker is a nucleic acid linker.

SEQ ID NO.20

SEQ ID NO.21

The invention also relates to a vector comprising a nucleic acid sequence as described herein. The invention also relates to a host cell comprising a nucleic acid sequence as described herein or a vector as described herein. The host cell may be a mammalian, bacterial or yeast cell.

The invention also relates to a kit comprising an immunoglobulin single variable domain or a pharmaceutical composition as described herein, a protein or a construct as described herein or a pharmaceutical composition as described herein and optionally instructions for use.

The single domain antibodies described herein can be obtained from a transgenic mammal, e.g., a rodent, that expresses an antibody having only a heavy chain upon stimulation with HSA antigen. Transgenic rodents, such as mice, preferably have reduced ability to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced ability to express endogenous light chain and/or heavy chain antibody genes. Rodents may therefore comprise modifications to disrupt the expression of endogenous kappa and lambda light chain and/or heavy chain antibody genes such that no functional light and/or heavy chains are produced, e.g., as explained further below.

One aspect also relates to a method for producing an antibody having only a human heavy chain or having a V capable of binding to HSA as described herein_HA method of binding a molecule to a domain, the method comprising

a) Immunizing a transgenic rodent, e.g., a mouse, with an HSA antigen, wherein the rodent expresses a nucleic acid construct comprising an unrearranged human heavy chain V gene and is incapable of producing a functional endogenous light or heavy chain,

b) human antibodies with only heavy chains were isolated.

Further steps may include, for example, generating a composition comprising V from the rodent (e.g., mouse)_HSequence libraries of domain sequences and isolation of V-containing polypeptides from the libraries_HSequence of Domain sequences isolation of V from the heavy chain-Only antibody_HA domain.

Another aspect also relates to methods for generating a single V capable of binding to human HSA_HA method of domain antibody, the method comprising

a) Immunizing a transgenic rodent, e.g., a mouse, with an HSA antigen, wherein the rodent expresses a nucleic acid construct comprising an unrearranged human heavy chain V gene and is incapable of producing a functional endogenous light or heavy chain,

b) generating a V comprising a protein from the rodent (e.g., mouse)_HA sequence library of domain sequences, and

c) isolation of a peptide comprising V from the library_HThe sequence of the domain sequence.

Further steps may include identifying a single V that binds to HSA_HDomain antibodies or antibodies with only heavy chains, for example by using the functional assays as shown in the examples.

Methods of using in vitro expression libraries to make or produce the polypeptides, nucleic acids, host cells, products, and compositions described herein may comprise the steps of:

a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences; and

b) screening said set, collection or library for amino acid sequences capable of binding/having affinity for HSA, and

c) isolating an amino acid sequence capable of binding/having affinity for HSA.

Also provided are methods for preparing the single V described herein_HA method of domain antibody, binding molecule or fusion protein, the method comprising culturing or maintaining a host cell as described herein under conditions such that the host cell produces or expresses a single V as described herein_HDomain antibodies, binding molecules or fusion proteins, and optionally further comprising isolating the single V so produced as described herein_HDomain antibodies, binding molecules or fusion proteins.

In the above methods, the set, collection or library of amino acid sequences can be displayed on a phage, phagemid, ribosome or suitable microorganism (e.g., yeast), for example, to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (groups, collections or libraries of) amino acid sequences will be clear to the skilled person (see e.g.phase Display of Peptides and Proteins: A Laboratory Manual, Academic Press; first edition (1996, 10/28) Brian K.Kay, Jill Winter, John McCafferty). Libraries, such as phage libraries, are prepared by isolating cells or tissues expressing antigen-specific, heavy chain-only antibodies, cloning V-encoding sequences from mRNA derived from the isolated cells or tissues_HThe sequence of the domain, and displaying the encoded protein using the library. V_HThe domains may be expressed in bacteria, yeast or other expression systems.

In various aspects and embodiments herein, the term rodent can refer to a mouse or a rat. In one embodiment, the rodent is a mouse. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, mice do not produce functional endogenous lambda light chains. In one embodiment, the lambda light chain locus is partially or completely deleted or rendered non-functional by insertion, inversion, recombination event, gene editing or gene silencing. For example, as described above, at least the constant region genes C1, C2, and C3 may be deleted or rendered non-functional by insertion or other modification. In one embodiment, the locus is functionally silenced such that the mouse does not produce a functional lambda light chain.

In addition, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, mice did not produce functional endogenous kappa light chains. In one embodiment, the kappa light chain locus is partially or completely deleted or rendered non-functional by insertion, inversion, recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced such that the mouse does not produce a functional kappa light chain.

For example, mice with functionally silenced endogenous lambda and kappa L-chain loci can be prepared as disclosed in WO2003/000737 (which is hereby incorporated by reference in its entirety).

Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus, for example as described in WO2004/076618 (herein incorporated by reference in its entirety). Thus, the mice did not produce functional endogenous heavy chains. In one embodiment, the heavy chain locus is partially or completely deleted or rendered non-functional by insertion, inversion, recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced such that the mouse does not produce a functional heavy chain.

In one embodiment, the mouse comprises a non-functional endogenous heavy chain locus, a non-functional endogenous lambda light chain locus, and a non-functional endogenous kappa light chain locus. Thus the mice do not produce any functional endogenous light or heavy chains. Thus, the mouse is a Triple Knockout (TKO) mouse.

The transgenic mice may comprise a vector, such as a Yeast Artificial Chromosome (YAC) for expression of a heterologous (preferably human) heavy chain locus. YACs are vectors that can be used to clone very large DNA inserts in yeast. Their ability to accept large DNA inserts, in addition to containing all three cis-acting structural elements essential for behavior on the native yeast chromosome (autonomously replicating sequences (ARS), Centromere (CEN) and two Telomeres (TEL)), allows them to reach the minimum size (150kb) required for chromosome-like stability in yeast cells and fidelity of delivery. The construction and use of YACs is well known in the art (e.g., Brusci, C.V., and Gjuracic, K.Yeast Art facial Chromosomes, Encyclopaedia of Life Sciences,2002 Macmillan Publishers Ltd, Nature Publishing Group).

For example, YACs may comprise an excess of unrearranged human VH, D, and J genes in combination with mouse immunoglobulin constant region genes, mouse enhancers, and regulatory regions that lack the CH1 domain. The human VH, D and J genes are human VH, D and J loci, and they are fully human unrearranged genes. YACs may be as described in WO 2016/062990.

Alternative methods known in the art can be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of human V, D and J genes in combination with mouse immunoglobulin constant region genes, mouse enhancers and regulatory regions that lack the CH1 domain.

Transgenic mice can be generated according to standard techniques shown in the examples. The two most characteristic routes for the generation of transgenic mice are by prokaryotic microinjection of genetic material into newly fertilized oocytes or by introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos. Regardless of how the genetic material is introduced, the manipulated embryo is transferred to a pseudopregnant female recipient, where pregnancy continues and candidate transgenic pups are produced.

The main difference between these broad approaches is that ES clones can be screened extensively before being used to generate transgenic animals. In contrast, prokaryotic microinjection relies on genetic material that is integrated into the host genome after introduction of the genetic material, and in general, successful integration of a transgene cannot be confirmed until after birth of the young.

Numerous methods are known in the art to assist and determine whether successful integration of a transgene has occurred. Transgenic animals can be generated by a variety of means, including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are a variety of tools and techniques available to drive and select for transgene integration and subsequent modification, including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques and nucleases to improve recombination efficiency. Most of these methods are commonly used to modify ES cells. However, several techniques are available to enhance transgenesis mediated by prokaryotic injection.

Further improvements can be used to generate transgenic lines more efficiently in a desired context. As described above, in preferred embodiments, endogenous mouse immunoglobulin expression is silenced to allow expression of heavy chain-only repertoire (reportiire) that can be exploited for drug discovery using only the introduced transgene. As described above, genetically manipulated mice can be used, such as TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain, and mouse lambda chain). Transfer of any introduced transgene to this TKO context can be achieved by breeding (conventional or including an IVF step) to provide efficient scaling of the process. However, TKO backgrounds may also be included in the transgenic process. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for transgenics.

Triple knockout mice that have a transgene introduced to express an immunoglobulin locus are referred to herein as TKO/Tg.

In one embodiment, the mouse is as described in WO 2016/062990. The invention also relates to rodents, preferably mice, that express the human heavy chain locus and have been immunized with the HSA antigen. The invention also relates to a rodent, preferably a mouse, as described above that expresses a heavy chain-only antibody comprising a human VH domain that binds to human HSA. Preferably, the rodent is unable to produce functional endogenous kappa and lambda light and/or heavy chains. The human heavy chain locus is located on a transgene as may be described above.

The invention also relates to antihuman HSA mono V_HA domain antibody or a heavy chain-only anti-human HSA antibody comprising human V_HThe domain is obtained or obtainable from a rodent, preferably a mouse, immunized with a human HSA antigen and expressing a human heavy chain locus. Preferably, the toothed gear is movableThe material was unable to produce functional endogenous kappa and lambda light and/or heavy chains. The human heavy chain locus is located on a transgene as may be described above.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter that is encompassed within the scope of the invention, including the methods of making and using the disclosure and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the disclosure. However, those skilled in the art will appreciate that the details of these examples should not be construed as limitations of the present invention, the scope of which should be construed from the claims appended to this disclosure and their equivalents. Various other aspects and embodiments of the disclosure will be apparent to those skilled in the art in view of this disclosure.

All documents mentioned in this specification are herein incorporated by reference in their entirety, including references to gene accession numbers, scientific publications, and references to patent publications.

The term "and/or" as used herein is considered to be a specific disclosure of each of the two specific features or components, with or without the other. For example, "a and/or B" shall be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed herein. Unless the context indicates otherwise, the description and definition of features set forth above is not limited to any particular aspect or embodiment of the invention, and applies equally to all aspects and embodiments described.

The invention is further illustrated in the following non-limiting examples.

Examples

Example 1 construction of Tg/TKO mice

Mice bearing germline-configured heavy chain antibody transgene loci in the context of silencing endogenous heavy and light chain antibody expression (triple knockout, or TKO) were generated as previously described (WO2004/076618 and WO2003/000737, Ren et al Genomics,84,686,2004; Zou et al, J.Immunol.,170,1354,2003). Briefly, transgenic mice were obtained following microinjection of newly fertilized oocytes with Yeast Artificial Chromosome (YAC) pronuclei containing excess human V in combination with mouse immunoglobulin constant region genes, mouse enhancers and regulatory regions that lack the CH1 domain_HD and J genes. Yeast Artificial Chromosomes (YACs) are vectors that can be used to clone very large DNA inserts in yeast. Their ability to accept large DNA inserts, in addition to containing all three cis-acting structural elements essential for behavior on the native yeast chromosome (autonomously replicating sequences (ARS), Centromere (CEN) and two Telomeres (TEL)), allows them to reach the minimum size (150kb) required for chromosome-like stability in yeast cells and fidelity of delivery. The construction and use OF YACs is well known in the art (e.g., Brusci, C.V., and Gjuracic, K.Yeast Industrial Chromosomes, ENCYCOPEDIA OF LIFE SCIENCES 2002Macmillan Publishers Ltd, Nature Publishing Group/www.els.net).

The YAC used was approximately 340kb, containing 10 natural configurations of human heavy chain V gene, human heavy chain D and J genes, murine Cgamma 1 gene and murine 3' enhancer gene. It lacks C_H1 exon. Specifically, YAC comprises (from 5 'to 3'): telomere-yeast TRP1 marker gene-centromere-23 personal V gene-human D gene-human J gene-mouse mu enhancer and switch mouse C gamma 1 (C)_H1. DELTA.) Gene-mouse 3' enhancer-hygromycin resistance Gene-Yeast marker gene HIS 3-telomeres.

Transgenic founder mice were backcrossed with animals lacking endogenous immunoglobulin expression to generate Tg/TKO lines for the immunization study.

Example 2 antigens for immunization

Serum purified human and cynomolgus serum albumin was used for immunization. Serum purified Human (HSA) and cynomolgus monkey (CSA) serum albumins were purchased from Sigma (cat No. a4327) and Abcam (cat No. ab 184894).

Example 3 immunization protocol

Three 8-12 week old crescindo mice each received a primary immunization with 50 μ g CSA, emulsified in complete freund's adjuvant and delivered subcutaneously, followed by 3 boosts of 10 μ g HSA, emulsified in incomplete freund's adjuvant, also administered subcutaneously, at weekly intervals following the initial priming. The final dose of HSA was administered intraperitoneally in phosphate buffered saline in the absence of adjuvant. At 49 days post primary immunization, mice were terminated and the arm and inguinal lymph nodes and spleen were collected into RNAlater (Qiagen catalog No. 76104). Serum was collected and stored for testing response.

Example 4 serum ELISA

Nunc Maxisorp plates were coated overnight at 4 ℃ with 1. mu.g/ml HSA in PBS solution. The plates were then washed with PBS supplemented with 0.05% tween 20, then PBS without tween, and blocked with 3% skim milk powder (Marvel) in PBS for at least one hour at room temperature. Serum dilutions in 3% Marvel/PBS were prepared in polypropylene tubes or plates and incubated at room temperature for at least one hour, then transferred to blocked ELISA plates where further incubation was performed for at least one hour. After washing in PBS/Tween and PBS, a solution of biotin-conjugated goat anti-mouse IgG, Fc γ subclass 1 specific antibody (Jackson 115-. Neutravidin-HRP solution (Pierce 31030) diluted 1:1000 in 3% Marvel/PBS was added to the ELISA plate and incubated for at least 30 minutes. After further washing, the ELISA was developed using TMB substrate (Sigma catalog No. T0440) and 10 minutes later by addition of 0.5M H₂SO₄The solution stops the reaction. The absorbance was determined by reading at 450 nm.

Example 5 Generation of libraries from immunized mice

a. Tissue treatment, RNA extraction and cDNA preparation

Spleen and lymph nodes from each immunized animal were collected in RNAlater. For each animal, 1/4 spleens and 4 lymph nodes were treated separately. Initially, the tissue is homogenized; followed by RNA precipitation. RNA purification was performed on qiapube using RNeasy 96 qiapube kit and qiapube HT plastics (plastics). Each RNA sample was then used to prepare cDNA using the Superscript III RT-PCR high fidelity kit.

b. Cloning into phagemid vectors

Cloning of V in the phagemid vector pUCG3 Using PCR-based methods_HA cDNA library.

Purifying V_HThe RT-PCR product was used as a giant primer with the linearized vector pUCG3-C-tag to provide a phagemid product for phage library creation. The PCR products were analyzed on a 1% agarose gel. Will V_HThe/phagemid PCR products were pooled by animal source and purified using Thermo GeneJet PCR purification kit according to the manufacturer's instructions. The eluted DNA was used to transform TG1 E.coli (Lucigen, Cat. No. 60502-2) by electroporation using Bio-Rad GenePulser Xcell.

The transformed 10-fold dilution series were plated on 2XTY agar plates with 2% (w/v) glucose and 100. mu.g/ml ampicillin. Colonies obtained on these plates were used to assess library size. The remainder of the transformation was plated on large 2XTY agar bioassay dishes supplemented with 2% (w/v) glucose and 100. mu.g/ml ampicillin. All agar plates were incubated overnight at 30 ℃. The library was harvested by adding 10ml of 2xTY liquid medium to a large bioassay dish. Bacterial colonies were gently scraped and OD600 recorded. After addition of an equal volume of 50% (v/v) glycerol solution, aliquots were stored in frozen vials at-80 ℃ or used directly in the phage selection process.

Example 6 isolation of V binding to HSA_HSelection strategy of

To generateLeader (lead), a PEG precipitated phage library from mice was used in panning selection with recombinant HSA protein conjugated to an immune tube (immuno-tube) at pH5 or pH7 to enrich for human serum albumin binding according to published methods (Antibody Engineering, edited by Benny Lo, chapter 8, p161-176,2004).

Example 7 assay for target binding

Screening for one or more of the following assays from different selectionsSelected V_HTo identify V binding to HSA and CSA_H。

a. Microsphere (bead) -based FLISA binding assay for human and cynomolgus serum albumin

For leader humobody V_HHSA194-D04 and HSA191E0-2, periplasmic extracts in plates in a homogeneous high-throughput assay using fluorescent micro-volume assay technique to identify specific clones that bind to human and cynomolgus serum albumin. The assay was performed using a fluorescent micro-volume assay technique which measures the cell-associated fluorescence within a defined volume at the bottom of an assay well in a homogeneous assay format (Dietz et al, Cytometry 23: 177-.

Small scale periplasmic bacterial extracts were prepared from 1ml of culture grown in deep well plates. The starting culture was used to inoculate a 96-well deep-well plate (Fisher, Cat. MPA-600-. When OD600 reached 0.5-1, V was induced by adding 100ul of 2XTY supplemented with IPTG (final concentration 5mM) and ampicillin_HProduction, and the culture at 30 degrees C under 220rpm shaking overnight growth. Coli were pelleted by centrifugation at 3200rpm for 10 minutes, and the cell pellet (pellet) from the supernatant was resuspended in 120. mu.l of ice-cold MES buffer (50mM MOPS, 0.5mM EDTA, 20% 0.5M sucrose), followed by addition of 180. mu.l of 1:5 diluted ice-cold extraction buffer. Cells were incubated on ice for 30 minutes and then centrifuged at 4500rpm for 15 minutes at 4 ℃. The supernatant was transferred to a polypropylene plate and incubated in 1xPBST blocking solution before direct use in ELISA.

A microsphere group: SOL-R4, SOL-R5 Tm carboxyl microspheres were coupled to recombinant HSA domain I-II (cat No. 9905), recombinant HSA domain II (cat No. 9902) and CSA. The microspheres were diluted to the desired concentration.

b. Purified V_HPreparation of

Using pJExpress vectorPurification of V from the supernatant of E.coli W3110_H. For this procedure, up to 1L of the culture was grown in 2XTY liquid medium (Melford, M2130) supplemented with 0.1% (w/v) glucose +50ug/ml kanamycin at 37 ℃ with shaking at 250 rpm. When OD600 reached 0.5-1, V was induced by addition of IPTG and kanamycin_HProduction, and the culture at 30 degrees C under 250rpm oscillation growth overnight. Coli were pelleted by centrifugation at 3200rpm, the resulting supernatant was harvested and V purified using the Capture Select C-tag XL affinity matrix (Thermo Fisher Cat. No. 2943072010)_HSize exclusion chromatography was then performed on the AKTA purification system using a HiLoad 26/600 superdraw 75pg column.

Spectrophotometric estimation of purified V_HAnd purity was evaluated using SDS PAGE.

c. Binding kinetics.

Binding studies were performed on human, cynomolgus and mouse serum albumin at pH7.4 and pH6.0 using single cycle kinetics on Biacore T200. Serum albumin (HSA, CSA, MSA) was coupled to CM5 chips by amine coupling using amine reagent coupling kit GE Healthcare BR-1000-50. The chip surface is activated according to the manufacturer's instructions. Serum albumin (HSA, CSA, MSA) was captured and cross-linked to the sensor chip surface by injection of serum albumin (HSA, CSA, MSA) in 10mM sodium acetate (ph 5.0). 1M ethanolamine was then injected to stabilize serum albumin on the surface. Through this procedure, approximately 300RU is fixed.

For kinetic measurements, four-fold serial dilutions of V in PBST pH7.4 or PBST pH6.0 were made_HSingle domain antibody (194D04-2 or 191E02-2) was injected at 25 ℃ at a flow rate of 45. mu.l/min for 120 seconds of binding and 300 seconds of dissociation. The binding response was corrected by subtracting the blank flow cell and the buffer run on the same flow cell. The traces were fitted using a 1:1 model.

Table 2:calculated humobody V_HKinetic constants with serum albumin and K_D。

Example 9-V_HSingle domain antibodies exhibit good stability

For purified V_HSize exclusion chromatography was performed. Briefly, purified V was purified_HStorage at 10mg/ml in selected buffers at 4 ℃ or 25 ℃ for 0-7 days, followed by analysis at different time points using a Waters H-Class Bio UPLC (detection at 280 nm) containing a PDA detector, at a Waters ACQUITY BEHAnd (4) separating on a SEC column. The sample was injected in a volume of 10. mu.l and run at a flow rate of 0.4 ml/min in a mobile phase containing 200mM NaCl, 100mM sodium phosphate, pH7.4 + 5% propan-1-ol. Data was collected for 6 minutes and the percentage of monomeric protein in the sample after storage was calculated.

After incubation at 4 ℃ and 40 ℃ for 7 days, no significant change was observed.

Table 3: stability of TPP-712 and 814. This shows the percentage of monomer present after 0,1 and 7 days.

Example 10 pharmacokinetic analysis of Single intravenous administration of half-life extending constructs in double transgenic humanized FcRn/HSA mice

Briefly, male or female GenOway Human HSA/FcRn Tg mice were dosed via the tail vein by single intravenous injections of the compounds listed in table 4 (n-3) at doses of 1 or 2 mg/kg. Some constructs comprise a purification/detection tag, such as: polyhistidine or FLAG tag. Blood samples were collected prior to dosing and at 0.083 hours, 1 hour, 8 hours, 24 hours, 48 hours, 72 hours, and 96 hours after administration of the saphenous vein drug. All animals were euthanized and blood collected 168 hours after dosing. Plasma was separated and stored at-80 ℃ until assayed. Plasma samples were analyzed on a Gyrolab immunoassay platform using biotinylated human PSMA or human CD137 as capture and human CD137Dylight650, human PD-1Dylight650 or anti-Flag-AF 647 rabbit monoclonal antibody (NEB, catalog No. 15009S) as detector. Data were analyzed using Gyros to obtain compound concentrations in plasma. Pharmacokinetic analysis of the data was done using PK solution 2.0(Excel insert). The results of the study show that in human HSA/FcRn Tg mice, the compounds have a half-life in the range of 19.9 to 78.7 hours when administered intravenously at 1 or 2 mg/kg.

TABLE 4 pK parameter summary table.

Example 8 PK Studies in cynomolgus monkeys

Prior to the start of cynomolgus PK studies, replacement, reduction and improvement considerations as well as ethical and scientific judgments (e.g., target expression/homology to humans, dose level, etc.) and risk were reviewed at the crescando Biologics and contract research institutes. The animal studies in this cynomolgus monkey PK study were conducted under the UK Home Office Project License (UK Home Office Project License) at a contract research institution located in the UK. The regulatory internal permit for this study strictly regulates the severity limit of the effects on the animals. The procedures in the protocol did not result in any effect exceeding the severity limits of the procedures.

Briefly, three male cynomolgus monkeys were dosed with the 4mg/kg Humabody construct (TPP-1246) listed in the table, and the Study was performed in Charles River Study. Serum samples were collected from all test subjects prior to dosing (-24 hours), 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 216 hours, 312 hours, 360 hours, 408 hours, and 504 hours after dosing, and frozen prior to testing. PK analysis was performed on serum samples using the assay developed by crescindo.

PK assay using a sandwich immunoassay format using the Gyrolab xpore immunoassay platform; the analytes (listed in the table) were immobilized by biotinylated CD137 antigen and detected by dyLight650 labeled PD-1. The assay was optimized and established to confirm range and reproducibility, and sample analysis was completed according to the established assay. PK data from the following time points were subjected to PK analysis: animal 01: 1 to 168 hours, animal 02: 1 to 120 hours and animal 03: from 1 to 168 hours. The PK parameters reported are the average of 3 individuals per outcome. T1/2 of TPP-1246 in cynomolgus monkey serum has been demonstrated to be 84.5 hours. + -. 7.58 hours. The data are shown in figure 1.

TABLE 5 estimation of PK parameters following single intravenous administration of TPP-1246 in cynomolgus monkeys

The molecules used in all the above experiments were based on the above molecules. Where appropriate, the molecule tested comprises a C-terminal sequence, such as a purification tag.