阅读说明：本技术 结合cd33、nkg2d和cd16的蛋白 (Proteins binding to CD33, NKG2D and CD16 ) 是由 格雷戈里·P·张 安·F·张 威廉·哈尼 布拉德利·M·伦德 比昂卡·普林茨 于 2018-02-20 设计创作，主要内容包括：描述了结合CD33、NKG2D受体和CD16的多特异性结合蛋白,以及用于治疗癌症的药物组合物和治疗方法。(Multispecific binding proteins that bind CD33, NKG2D receptor, and CD16 are described, as well as pharmaceutical compositions and methods of treatment for treating cancer.)

1. A protein, comprising:

(a) A first antigen binding site that binds NKG 2D;

(b) A second antigen binding site that binds CD 33; and

(c) An antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site of CD 16.

2. The protein of claim 1, wherein the first antigen-binding site binds to NKG2D in humans, non-human primates and rodents.

3. The protein of claim 1 or 2, wherein the first antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

4. The protein of claim 3, wherein the heavy chain variable domain and the light chain variable domain are present on the same polypeptide.

5. The protein of any one of claims 3-4, wherein the second antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

6. The protein of claim 5, wherein the heavy chain variable domain and the light chain variable domain of the second antigen binding site are present on the same polypeptide.

7. The protein of claim 5 or 6, wherein the light chain variable domain of the first antigen binding site has an amino acid sequence that is identical to the amino acid sequence of the light chain variable domain of the second antigen binding site.

8. The protein of any one of the preceding claims, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID No. 1.

9. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO 41 and a light chain variable domain that is at least 90% identical to SEQ ID NO 42.

10. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID No. 43 and a light chain variable domain that is at least 90% identical to SEQ ID No. 44.

11. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO 45 and a light chain variable domain that is at least 90% identical to SEQ ID NO 46.

12. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO 47 and a light chain variable domain that is at least 90% identical to SEQ ID NO 48.

13. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO:69 and a light chain variable domain that is at least 90% identical to SEQ ID NO: 70.

14. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID No. 77 and a light chain variable domain that is at least 90% identical to SEQ ID No. 78.

15. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID NO 85 and a light chain variable domain that is at least 90% identical to SEQ ID NO 86.

16. The protein of any one of claims 1-7, wherein the first antigen binding site comprises a heavy chain variable domain that is at least 90% identical to SEQ ID No. 133 and a light chain variable domain that is at least 90% identical to SEQ ID No. 134.

17. the protein of claim 1 or 2, wherein the first antigen binding site is a single domain antibody.

18. The protein of claim 17, wherein the single domain antibody is a V_HH fragment or V_NARAnd (3) fragment.

19. The protein of any one of claims 1-2 or 17-18, wherein the second antigen binding site comprises a heavy chain variable domain and a light chain variable domain.

20. The protein of claim 19, wherein the heavy chain variable domain and the light chain variable domain of the second antigen binding site are present on the same polypeptide.

21. The protein of any one of the preceding claims, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 93 and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 94.

22. The protein of any one of claims 1-20, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 101 and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 102.

23. The protein of any one of claims 1-20, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 109 and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 110.

24. The protein of any one of claims 1-20, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 117 and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 118.

25. The protein of any one of claims 1-20, wherein the heavy chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 125 and the light chain variable domain of the second antigen binding site comprises an amino acid sequence at least 90% identical to SEQ ID No. 126.

26. The protein of any one of claims 1-4 or 8-16, wherein the second antigen binding site is a single domain antibody.

27. the protein of claim 26, wherein the second antigen binding site is V_HH fragment or V_NARAnd (3) fragment.

28. The protein of any one of the preceding claims, wherein the protein comprises a portion of an antibody Fc domain sufficient to bind CD16, wherein the antibody Fc domain comprises a hinge and a CH2 domain.

29. The protein of claim 28, wherein the antibody Fc domain comprises the hinge and CH2 domains of a human IgG1 antibody.

30. The protein of claim 28 or 29, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to amino acid 234-332 of the human IgG1 antibody.

31. The protein of any one of claims 28-30, wherein the Fc domain comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs at one or more positions selected from the group consisting of: q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, K439.

32. A formulation comprising the protein of any one of the preceding claims and a pharmaceutically acceptable carrier.

33. A cell comprising one or more nucleic acids expressing a protein according to any one of claims 1-31.

34. A method of directly and/or indirectly enhancing tumor cell death, the method comprising exposing a tumor and a natural killer cell to a protein of any one of claims 1-31.

35. A method of treating cancer, wherein the method comprises administering to a patient a protein according to any one of claims 1-31 or a formulation according to claim 32.

36. The method of claim 35, wherein the cancer is selected from AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast phase of chronic myelogenous leukemia, and ALL.

Technical Field

The present invention relates to multispecific binding proteins that bind to CD33, NKG2D receptor, and CD 16.

Background

Despite the large research efforts and scientific advances reported in the literature for the treatment of this disease, cancer remains an important health problem. Some of the most commonly diagnosed cancers in adults include prostate, breast and lung cancer. Hematological malignancies have a lower frequency than solid cancers, but have a lower survival rate. Current treatment options for these cancers are not effective in all patients and/or may have significant adverse side effects. Treatment of other types of cancer using existing treatment options remains challenging.

Cancer immunotherapy are desirable because they are highly specific and can utilize the patient's own immune system to promote destruction of cancer cells. Fusion proteins, such as bispecific T cell engagers (engage) are cancer immunotherapies described in the literature that bind to tumor cells and T cells to promote destruction of tumor cells. Antibodies that bind to certain tumor-associated antigens and certain immune cells have been described in the literature. See, for example, WO 2016/134371 and WO 2015/095412.

Natural Killer (NK) cells are a component of the innate immune system and account for about 15% of circulating lymphocytes. NK cells penetrate almost all tissues and were originally characterized by their ability to kill tumor cells efficiently without prior sensitization. Activated NK cells kill target cells in a manner similar to cytotoxic T cells-i.e. by cytolytic granules containing perforin and granzyme and by the death receptor pathway. Activated NK cells also secrete inflammatory cytokines, such as IFN- γ and chemokines, which promote the recruitment of other leukocytes to the target tissue.

NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited by activating killer immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated by their activation receptors (e.g., NKG2D, NCR, DNAM 1). NK cells are also activated by the constant regions of some immunoglobulins through the CD16 receptor on their surface. The overall sensitivity of NK cells to activation depends on the sum of the stimulatory and inhibitory signals.

CD33 is a member of sialic acid binding immunoglobulin-like lectins. As a transmembrane receptor expressed predominantly on cells of the myeloid lineage, CD33 modulates inflammation and immune responses through inhibition of the tyrosine kinase-driven signaling pathway. For example, CD33 has been shown to constitutively inhibit the production of proinflammatory cytokines such as IL-1 β, TNF- α, and IL-8 by human monocytes.

CD33 is associated with hematopoietic cancers. It is widely expressed in the naive cells of almost all Acute Myeloid Leukemia (AML). Furthermore, hematopoietic cancer stem and/or progenitor cells were found to be CD33⁺This means that CD 33-directed therapy can in this case potentially eradicate malignant stem and/or progenitor cells, while retaining normal hematopoietic stem cells. In addition to expression in AML, CD33 is also present in other myeloid tumors (e.g., myelodysplastic syndrome and myeloproliferative tumors) and subpopulations of B-cell and T-cell Acute Lymphoblastic Leukemia (ALL)/lymphoblastic lymphoma. This pattern of expression results in the use of CD 33-directed therapeutics in patients with malignancies including AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast phase of chronic myelogenous leukemia, and ALL.

Summary of The Invention

The present invention provides multispecific binding proteins that bind to CD33 on cancer cells and to the NKG2D receptor and CD16 receptor on natural killer cells. Such proteins may bind to more than one NK-activating receptor and may block the binding of natural ligands to NKG 2D. In certain embodiments, the protein may agonize NK cells in humans and other species, such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in further detail below.

Accordingly, one aspect of the present invention provides a protein comprising a first antigen binding site that binds NKG 2D; (ii) a second antigen binding site that binds to CD 33; and an antibody Fc domain, a portion of which is sufficient to bind CD16, or to bind a third antigen binding site of CD 16. The antigen binding sites may each comprise an antibody heavy chain variable domain and an antibody light chain variable domain, e.g., as arranged in an antibody, or fused together to form an scFv, or one or more of the antigen binding sites may be a single domain antibody, e.g., a V_HH antibodies, e.g. camelid antibodies, or V_NARAntibodies, such as those found in cartilaginous fish.

In one embodiment, the first antigen binding site that binds to NKG2D may comprise a heavy chain variable domain related to SEQ ID NO:1, e.g., by having an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:1, and/or comprises an amino acid sequence that is identical to the CDR1(SEQ ID NO:54), CDR2(SEQ ID NO:55), and CDR3(SEQ ID NO:56) sequences of SEQ ID NO: 1. Alternatively, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO. 41 and a light chain variable domain associated with SEQ ID NO. 42. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 41 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:57), CDR2(SEQ ID NO:58) and CDR3(SEQ ID NO:59) sequences of SEQ ID NO 41. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 42 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:60), CDR2(SEQ ID NO:61) and CDR3(SEQ ID NO:62) sequences of SEQ ID NO 42. In other embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO 43 and a light chain variable domain associated with SEQ ID NO 44. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 43 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:63), CDR2(SEQ ID NO:64) and CDR3(SEQ ID NO:65) sequences of SEQ ID NO 43. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 44 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:66), CDR2(SEQ ID NO:67) and CDR3(SEQ ID NO:68) sequences of SEQ ID NO 44.

In some embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID No. 45 and a light chain variable domain associated with SEQ ID No. 46, for example by making the amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 45 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID No. 46, respectively. In another embodiment, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO. 47 and a light chain variable domain associated with SEQ ID NO. 48, e.g., by having an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO. 47 and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO. 48, respectively.

In some embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO:69 and a light chain variable domain associated with SEQ ID NO: 70. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:69 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:71), CDR2(SEQ ID NO:72) and CDR3(SEQ ID NO:73) sequences of SEQ ID NO: 69. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 70 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:74), CDR2(SEQ ID NO:75) and CDR3(SEQ ID NO:76) sequences of SEQ ID NO 70. In some embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO 77 and a light chain variable domain associated with SEQ ID NO 78. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 77 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:69), CDR2(SEQ ID NO:80) and CDR3(SEQ ID NO:81) sequences of SEQ ID NO 77. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:78 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:82), CDR2(SEQ ID NO:83) and CDR3(SEQ ID NO:84) sequences of SEQ ID NO: 78.

In some embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO. 85 and a light chain variable domain associated with SEQ ID NO. 86. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 85 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:87), CDR2(SEQ ID NO:88) and CDR3(SEQ ID NO:89) sequences of SEQ ID NO 85. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 86 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:90), CDR2(SEQ ID NO:91) and CDR3(SEQ ID NO:92) sequences of SEQ ID NO 86.

In some embodiments, the first antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO. 133 and a light chain variable domain associated with SEQ ID NO. 134. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 133 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:135), CDR2(SEQ ID NO:136) and CDR3(SEQ ID NO:137) sequences of SEQ ID NO 133. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 134 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:138), CDR2(SEQ ID NO:139) and CDR3(SEQ ID NO:140) sequences of SEQ ID NO 134.

The second antigen binding site can optionally comprise a heavy chain variable domain associated with SEQ ID NO 93 and a light chain variable domain associated with SEQ ID NO 94. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID No. 93 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:95), CDR2(SEQ ID NO:96) and CDR3(SEQ ID NO:97) sequences of SEQ ID No. 93. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 94 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:98), CDR2(SEQ ID NO:99) and CDR3(SEQ ID NO:100) sequences of SEQ ID NO 94.

Alternatively, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO 101 and a light chain variable domain associated with SEQ ID NO 102. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 101 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:103), CDR2(SEQ ID NO:104) and CDR3(SEQ ID NO:105) sequences of SEQ ID NO 101. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 58 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:106), CDR2(SEQ ID NO:107) and CDR3(SEQ ID NO:108) sequences of SEQ ID NO 102.

In another embodiment, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO. 109 and a light chain variable domain associated with SEQ ID NO. 110. For example, the heavy chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 59 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:111), CDR2(SEQ ID NO:112) and CDR3(SEQ ID NO:113) sequences of SEQ ID NO 109. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 110 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:114), CDR2(SEQ ID NO:115) and CDR3(SEQ ID NO:116) sequences of SEQ ID NO 110.

In another embodiment, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO 117 and a light chain variable domain associated with SEQ ID NO 118. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO:117 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:119), CDR2(SEQ ID NO:120) and CDR3(SEQ ID NO:121) sequences of SEQ ID NO: 117. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 118 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:122), CDR2(SEQ ID NO:123) and CDR3(SEQ ID NO:124) sequences of SEQ ID NO 118.

In another embodiment, the second antigen binding site may comprise a heavy chain variable domain associated with SEQ ID NO 125 and a light chain variable domain associated with SEQ ID NO 126. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID No. 125 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:127), CDR2(SEQ ID NO:128) and CDR3(SEQ ID NO:129) sequences of SEQ ID No. 125. Similarly, the light chain variable domain of the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO 126 and/or comprise an amino acid sequence identical to the CDR1(SEQ ID NO:130), CDR2(SEQ ID NO:131) and CDR3(SEQ ID NO:132) sequences of SEQ ID NO 126.

In some embodiments, the second antigen-binding site comprises a light chain variable domain that has an amino acid sequence that is identical to the amino acid sequence of the light chain variable domain present in the first antigen-binding site.

In some embodiments, the protein comprises a portion of an antibody Fc domain sufficient to bind CD16, wherein the antibody Fc domain comprises a hinge and a CH2 domain, and/or an amino acid sequence that is at least 90% identical to amino acid sequence 234-332 of a human IgG antibody.

Also provided are formulations comprising one of these proteins; cells comprising one or more nucleic acids expressing these proteins, and methods of using these proteins to enhance tumor cell death.

Another aspect of the invention provides a method of treating cancer in a patient. The method comprises administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein. Exemplary cancers for treatment using this multispecific binding protein include, for example, wherein the cancer is selected from AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast phase of chronic myelogenous leukemia, and ALL.

Brief description of the drawings

FIG. 1 is a schematic representation of a heterodimeric multispecific antibody. Each arm may represent either an NKG2D binding domain or a CD33 binding domain. In some embodiments, the NKG2D and CD33 binding domains may share a common light chain.

FIG. 2 is a schematic representation of a heterodimeric multispecific antibody. The NKG2D or CD33 binding domain may be in scFv format (right arm).

Figure 3 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to human recombinant NKG2D in an ELISA assay.

Figure 4 is a line graph showing the binding affinity of NKG2D binding domain (listed as a clone) to cynomolgus monkey recombinant NKG2D in an ELISA assay.

Figure 5 is a line graph showing the binding affinity of NKG2D binding domains (listed as clones) to mouse recombinant NKG2D in an ELISA assay.

Figure 6 is a bar graph showing the binding of NKG2D binding domains (listed as clones) by flow cytometry to EL4 cells expressing human NKG2D, showing fold Mean Fluorescence Intensity (MFI) against background.

Figure 7 is a bar graph showing the binding of NKG2D binding domains (listed as clones) by flow cytometry to EL4 cells expressing mouse NKG2D, showing fold Mean Fluorescence Intensity (MFI) against background.

FIG. 8 is a line graph showing the specific binding affinity of NKG2D binding domain (listed as a clone) to recombinant human NKG2D-Fc by competition with the natural ligand ULBP-6.

FIG. 9 is a line graph showing the specific binding affinity of the NKG2D binding domain (listed as a clone) to recombinant human NKG2D-Fc by competition with the natural ligand MICA.

FIG. 10 is a line graph showing the specific binding affinity of the NKG2D binding domain (listed as a clone) to recombinant mouse NKG2D-Fc by competition with the natural ligand Rae-1 delta.

Figure 11 is a bar graph showing activation of human NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF-alpha positive cells expressing human NKG2D-CD3 zeta fusion protein.

FIG. 12 is a bar graph showing the activation of mouse NKG2D by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF- α positive cells expressing mouse NKG2D-CD3 ζ fusion protein.

Figure 13 is a bar graph showing activation of NKG2D binding domains (listed as clones) on human NK cells.

Figure 14 is a bar graph showing activation of NKG2D binding domains (listed as clones) on human NK cells.

Figure 15 is a bar graph showing activation of mouse NK cells by the NKG2D binding domain (listed as a clone).

Figure 16 is a bar graph showing activation of mouse NK cells by the NKG2D binding domain (listed as a clone).

FIG. 17 is a bar graph showing the cytotoxic effect of the NKG2D binding domain (listed as a clone) on tumor cells.

Figure 18 is a bar graph showing the melting temperature of NKG2D binding domains (listed as clones) measured by differential scanning fluorimetry.

FIGS. 19A-19C are bar graphs of synergistic activation of NK cells using CD16 and NKG2D binding. FIG. 19A shows the level of CD107 a; figure 19B shows levels of IFN γ; figure 19C shows CD107a and IFN γ levels. The graph shows the mean (n ═ 2) ± SD. Data represent five independent experiments performed using five different healthy donors.

Figure 20 is a schematic representation of trinkets in the form of trifunctional antibodies (Triomab), which are trifunctional bispecific antibodies that retain an IgG-like shape. The chimera consists of two half-antibodies derived from two parent antibodies, each half-antibody having one light chain and one heavy chain. The trifunctional antibody format may be a heterodimeric construct comprising a rat antibody of 1/2 and a mouse antibody of 1/2.

FIG. 21 is a schematic representation of TriNKET in the form of a KiH common Light Chain (LC) involving a knob-and-hole (KIH) technique. KiH is a heterodimer comprising 2 fabs that bind to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. The kirk form of TriNKET may be a heterodimeric construct with 2 fabs binding to targets 1 and 2, comprising two different heavy chains and a common light chain pairing with the two heavy chains.

FIG. 22 is a dual variable domain immunoglobulin (DVD-Ig)^TM) Schematic representation of a format of TriNKET that combines the target binding domains of two monoclonal antibodies by a flexible, naturally occurring linker and produces a tetravalent IgG-like molecule. DVD-Ig^TMIs a homodimer construct in which the variable domain of the Fab targeting antigen 2 is fused to the N-terminus of the variable domain of the Fab targeting antigen 1. The construct comprises normal Fc.

Figure 23 is a schematic representation of TriNKET in the form of an Orthogonal Fab interface (Ortho-Fab), a Fab heterodimer construct comprising 2 binding to target 1 and target 2 fused to Fc. LC-HC pairing is ensured by an orthogonal interface. Heterodimerization is ensured by mutations in the Fc.

Figure 24 is a schematic representation of TrinKET in 2-in-1 Ig form.

Figure 25 is a schematic representation of ES form of TriNKET, a heterodimeric construct comprising 2 different fabs binding to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc.

Figure 26 is a schematic representation of the Fab arm exchanged form of TriNKET: antibodies bispecific antibodies are generated by exchanging Fab arms by replacing the heavy chain and attached light chain (half-molecules) with a heavy chain-light chain pair from another molecule. The Fab arm exchange format (cFae) is a heterodimer comprising 2 fabs that bind to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

Figure 27 is a schematic representation of TriNKET in the form of SEED, a heterodimer comprising 2 fabs binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations.

FIG. 28 is a schematic representation of the LuZ-Y form of TriNKET, in which a leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer comprising two different scfabs that bind to targets 1 and 2 fused to an Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of the Fc.

FIG. 29 is a schematic representation of TriNKET in the form of a Cov-X-body.

Figures 30A-30B are schematic representations of the κ λ -body form of TriNKET, a heterodimeric construct with two different fabs fused to an Fc stabilized by heterodimerization mutations: the Fab1 targeting antigen 1 contained kappa LC, while the second Fab targeting antigen 2 contained lambda LC. FIG. 30A is an exemplary illustration of a κ λ -body form; FIG. 30B is an exemplary illustration of another κ λ -body.

Figure 31 is an Oasc-Fab heterodimer construct comprising a Fab binding to target 1 and a scFab binding to target 2 fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

Figure 32 is DuetMab, a heterodimer construct comprising two different fabs that bind to antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fab1 and 2 contain different S-S bridges which ensure correct Light Chain (LC) and Heavy Chain (HC) pairing.

Figure 33 is a crosssmab, a heterodimer construct with two different fabs binding to targets 1 and 2 fused to Fc stabilized by heterodimerization. The CL and CH1 domains are exchanged with the VH and VL domains, e.g., CH1 is fused in-line with VL and CL is fused in-line with VH.

FIG. 34 is Fit-Ig, which is a homodimer construct in which Fab binding to antigen 2 is fused to the N-terminus of the HC of Fab binding to antigen 1. The construct comprises a wild-type Fc.

Fig. 35A-35B are binding profiles of TriNKET targeting CD33 with NKG2D expressed on EL4 cells. Figure 35A shows binding of trinkets compared to monoclonal antibodies comprising the corresponding NKG2D binding domains. Figure 35B shows the binding profile of CD 33-targeted TriNKET comprising 6 different NKG2D binding domains.

Fig. 36A and 36B are binding profiles of TriNKET targeting CD33 to CD33 expressed on MV4-11 human AML cells. Fig. 36C is a binding profile of TriNKET and CD33 monoclonal antibodies targeting CD33 to CD33 expressed on Molm-13 human AML cells. Figure 36D is a binding profile of TriNKET and CD33 monoclonal antibodies targeting CD33 to CD33 expressed on human AML cell line MV 4-11.

FIGS. 37A-37B are line graphs showing TriNKET-mediated activation of quiescent or IL-2 activated human NK cells co-cultured with the CD 33-expressing human AML cell line MV 4-11. Figure 37A shows the TriNKET-mediated activation of quiescent human NK cells. Figure 37B shows the TriNKET-mediated activation of IL-2 activated human NK cells from the same donor. NK cells alone, NK cells co-cultured with MV4-11 cells but without TriNKET, and TriNKET targeted to CD20 were used as controls.

FIGS. 38A-38C are histograms showing expression of high affinity FcR γ I (CD64) on three human AML cell lines, i.e., the Molm-13 cell line (FIG. 38A), the MV4-11 cell line (FIG. 38B), and the THP-1 cell line (FIG. 38C).

FIGS. 39A-39B are line graphs of CD33 monoclonal antibody or TriNKET-mediated activation of human NK cells co-cultured with Molm-13 (FIG. 39B) or THP-1 (FIG. 39A) cells. Figure 39C shows activation of human NK cells by TriNKET co-cultured with MV4-11 human AML cell line. HER2-TriNKET was used as a control.

Figures 40A-40C are line graphs of human NK cytotoxicity on three human AML cell lines mediated by TriNKET targeting CD33 and the corresponding CD33 monoclonal antibody. FIG. 40A shows that the CD33 monoclonal antibody shows reduced efficacy against MV4-11 cells, which express CD64 but at lower levels than THP-1. FIG. 40B shows that the CD33 monoclonal antibody shows good efficacy against Molm-13 cells that do not express CD 64. FIG. 40C shows that the CD33 monoclonal antibody has no effect on THP-1 cells.

FIG. 41 shows TriNKET-mediated cytotoxicity of quiescent human NK cells against Molm-13 cells.

Fig. 42A is a bar graph showing that B cells from healthy donors were protected from trinketet-mediated lysis targeting CD 33. Fig. 42B is a bar graph showing that autologous CD33+ myeloid lineage cells are protected from TriNKET-mediated NK cell responses targeting CD33, and thus are resistant to TriNKET-mediated lysis.

Detailed description of the invention

The present invention provides multispecific binding proteins that bind CD33 on cancer cells and NKG2D receptors and CD16 receptors on natural killer cells to activate natural killer cells, pharmaceutical compositions comprising such multispecific binding proteins, and therapeutic methods using such multispecific proteins and pharmaceutical compositions, including for the treatment of cancer. Various aspects of the invention are set forth in sections below; however, aspects of the invention described in one particular section are not limited to any particular section.

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

The terms "a" and "an" as used herein mean "one or more" and include the plural unless the context does not otherwise apply.

As used herein, the term "antigen binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and light chain ("L"). The three highly divergent segments within the V regions of the heavy and light chains are called "hypervariable regions" which are interposed between more conserved flanking segments called "framework regions" or "FRs". Thus, the term "FR" refers to amino acid sequences that naturally occur between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". In certain animals, such as camels and cartilaginous fish, the antigen binding site is formed by a single antibody chain providing a "single domain antibody". The antigen binding site may be present in an intact antibody, in an antigen binding fragment of an antibody that retains an antigen binding surface, or in a recombinant polypeptide such as an scFv that links a heavy chain variable domain to a light chain variable domain in a single polypeptide using a peptide linker.

The term "tumor associated antigen" as used herein refers to any antigen, including but not limited to proteins, glycoproteins, gangliosides, carbohydrates, lipids associated with cancer. Such antigens may be expressed on malignant tumor cells or in the tumor microenvironment, such as on tumor-associated vessels, extracellular matrix, mesenchymal matrix or immune infiltrates.

As used herein, the terms "subject" and "patient" refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, equine, bovine, porcine, canine, feline, etc.), and more preferably include humans.

As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the invention) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications or administrations, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, e.g., reduction, modulation, amelioration, or elimination, that results in the amelioration of a condition, disease, disorder, etc., or amelioration of a symptom thereof.

As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15 th edition, Mack pub.

As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention that is capable of providing a compound of the present invention or an active metabolite or residue thereof upon administration to a subject. As known to those skilled in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not per se pharmaceutically acceptable, may be used in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW₄ ⁺Wherein W is C_1-4Alkyl, and the like.

Exemplary salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include salts with suitable cations such as Na⁺、NH₄ ⁺And NW₄ ⁺(wherein W is C_1-4Alkyl) and the like.

For therapeutic use, salts of the compounds of the present invention are expected to be pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

Throughout the specification, when a composition is described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is contemplated that there will additionally be compositions of the present invention consisting essentially of, or consisting of, the recited components, and that there will be processes and methods according to the present invention consisting essentially of, or consisting of, the recited process steps.

Generally, the specified percentages of the composition are by weight unless otherwise indicated. Furthermore, if the variable is not concomitantly defined, the previous definition of the variable is dominant.

I. Protein

The present invention provides multispecific binding proteins that bind CD33 on cancer cells and NKG2D receptors and CD16 receptors on natural killer cells to activate natural killer cells. The multispecific binding proteins may be used in pharmaceutical compositions and methods of treatment described herein. The binding of multispecific binding proteins to NKG2D receptors and CD16 receptors on natural killer cells enhances the cancer cell destroying activity of natural killer cells. The binding of the multispecific binding protein to CD33 on cancer cells brings the cancer cells in proximity to natural killer cells, which facilitates the direct and indirect destruction of cancer cells by natural killer cells. Further description of exemplary multispecific binding proteins is provided below.

The first component of the multispecific binding protein binds to cells expressing the NKG2D receptor, which may include, but is not limited to, NK cells, γ δ T cells, and CD8⁺α β T cells. Upon NKG2D binding, multispecific binding proteins may block natural ligands (e.g., ULBP6 and MICA) from binding to NKG2D and activating the NKG2D receptor.

The second component of the multispecific binding protein binds to cells expressing CD33, which may include, but is not limited to, cells that may include, but may not be limited to, AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast phase of chronic myelogenous leukemia, and ALL.

The third component of the multispecific binding protein binds to cells expressing CD16, CD16 is an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

The multispecific binding proteins described herein may take several forms. For example, one form is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain (fig. 1). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH 2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain comprises a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain and the first immunoglobulin heavy chain together form an antigen binding site that binds NKG 2D. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH 2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. The second immunoglobulin light chain comprises a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain and the second immunoglobulin heavy chain together form an antigen binding site that binds CD 33. Together, the first Fc domain and the second Fc domain are capable of binding to CD16 (fig. 1). In some embodiments, the first immunoglobulin light chain may be identical to the second immunoglobulin light chain.

Another exemplary form relates to a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (fig. 2). The first immunoglobulin heavy chain comprises a first Fc (hinge-CH 2-CH3) domain, which is fused by a linker or antibody hinge to a single chain variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain paired with and binding to NKG2D or CD 33. The second immunoglobulin heavy chain comprises a second Fc (hinge-CH 2-CH3) domain, a second heavy chain variable domain, and optionally a CH1 heavy chain domain. Immunoglobulin light chains comprise a light chain variable domain and a constant light chain domain. The second immunoglobulin heavy chain is paired with an immunoglobulin light chain and binds to NKG2D or CD 33. The first Fc domain and the second Fc domain are capable of binding together to CD16 (fig. 2).

One or more additional binding motifs may be fused to the C-terminus of the constant region CH3 domain, optionally linked by a linker sequence. In certain embodiments, the antigen binding site may be a single chain or disulfide stabilized variable region (scFv) or may form a tetravalent or trivalent molecule.

In some embodiments, the multispecific binding protein is in the form of a trifunctional antibody (Triomab), which is a trifunctional bispecific antibody that retains an IgG-like shape. The chimera consists of two half-antibodies derived from two parent antibodies, each half-antibody having one light chain and one heavy chain.

In some embodiments, the multispecific binding protein is in the form of a KiH common Light Chain (LC), which involves a knob and hole (KiH) technique. KIH relates to engineered C_H3 domains to create a "knob" or "hole" in each heavy chain to promote heterodimerization. The concept behind the "Knob (KiH)" Fc technique is to introduce a "knob" (e.g., T366W in EU numbering) in one CH3 domain (CH3A) by replacing small residues with bulky residues_CH3A). To accommodate a "pestle", a complementary "hole" surface (e.g., T366S/L368A/Y407V) is created on the other CH3 domain (CH3B) by replacing the nearest neighbor with a smaller residue_CH3B). Optimization of "hole" mutations by structural-directed phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P.Stable heterologous analogs from the domain interface of host computer using a phase display library J.mol.biol. (1997)270(1): 26-35). The X-ray crystal structure of the KiHFc variant (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C et al, anticancer compatibility of knob and hole aggregation of half-antibodies homo modems amplified by a CH2-CH3 hydrophic interaction. J.mol.biol. (2014)426(9): 1947-57; Mimof, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. crystal structure of a novel enzymological engineering dimerization of Fc variable after modification in favor of the inter-domain thermodynamic interface of the hydrophobic molecules (2014) 58-358) and the inter-domain thermodynamic interface of the molecule dimerization of the molecule (2014) 58-358Disruption of favorable interactions is prevented against homodimerization.

In some embodiments, the multispecific binding protein is a double variable domain immunoglobulin (DVD-Ig)^TM) A format that combines the target binding domains of two monoclonal antibodies by a flexible, naturally occurring linker and produces a tetravalent IgG-like molecule.

In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab). In the ortho-Fab IgG method (Lewis SM, Wu X, Pustlnik A, Sereno A, Huang F, Rick HL et al. Generation of bispecific IgG antibodies by structure-based design of orthogonal Fab interface. Nat. Biotechnol. (2014)32(2): 191-8), the structure-based region design is in LC and HC in only one Fab_VH-CH1Complementary mutations were introduced at the interface without any change to the other fabs.

In some embodiments, the multispecific binding protein is a 2-in-1 Ig form. In some embodiments, the multispecific binding protein is in the form of ES, which is a heterodimeric construct comprising 2 different fabs that bind to target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations in the Fc. In some embodiments, the multispecific binding protein is in the form of a κ λ -body, which is a heterodimeric construct with two different fabs fused to an Fc stabilized by heterodimerization mutations: the Fab1 targeting antigen 1 contained kappa LC, while the second Fab targeting antigen 2 contained lambda LC. FIG. 30A is an exemplary illustration of a κ λ -body form; FIG. 30B is an exemplary illustration of another κ λ -body.

In some embodiments, the multispecific binding protein is in the form of a Fab arm exchange (an antibody exchanges Fab arms by exchanging a heavy chain and an attached light chain (half molecule) with a heavy chain-light chain pair from another molecule, resulting in a bispecific antibody). In some embodiments, the multispecific binding protein is in the form of a SEED body. The ability of the chain exchange engineered domain (SEED) platform to be designed for the generation of asymmetric and bispecific antibody-like molecules extends the therapeutic applications of natural antibodies. The protein engineering platform is based on the exchange of structurally related immunoglobulin sequences within the conserved CH3 domain. The SEED design allows efficient production of AG/GA heterodimers while disfavoring homodimerization of AG and GA SEED CH3 domains. (Mudam. et al, Protein Eng. Des. sel. (2011,24(5): 447-54)). In some embodiments, the multispecific binding protein is in the LuZ-Y form, wherein a leucine zipper is used to induce heterodimerization of two different HCs (Wranik, BJ. et al, j.biol.chem. (2012),287: 43331-9).

In some embodiments, the multispecific binding protein is in the form of a Cov-X-body. In the bispecific CovX-body, two different peptides are linked together using a branched azetidinone linker and fused to a scaffold antibody in a site-specific manner under mild conditions. Despite the association of pharmacophores with functional activity, antibody scaffolds confer long half-life and Ig-like distribution. The pharmacophore can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies. (Dopplapaudi VR et al, PNAS (2010),107 (52); 22611-22616).

In some embodiments, the multispecific binding protein is in the form of an Oasc-Fab heterodimer comprising a Fab that binds to target 1 and a scFab that binds to target 2 fused to an Fc. Heterodimerization is ensured by mutations in the Fc.

In some embodiments, the multispecific binding protein is in the form of a DuetMab, a heterodimeric construct comprising two different fabs that bind to antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fab1 and 2 contain different S-S bridges, which ensure correct LC and HC pairing.

In some embodiments, the multispecific binding protein is in the form of a CrossmAb, a heterodimeric construct with two different fabs that bind to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains are exchanged with the VH and VL domains, e.g., CH1 is fused in-line with VL and CL is fused in-line with VH.

In some embodiments, the multispecific binding protein is a Fit-Ig form that is a homodimeric construct in which a Fab that binds antigen 2 is fused to the N-terminus of the HC of a Fab that binds antigen 1. The construct comprises a wild-type Fc.

Table 1 lists the peptide sequences of the heavy and light chain variable domains, which in combination can bind to NKG 2D. The NKG2D binding domains may differ in their binding affinity to NKG2D, although they all activate human NKG2D and NK cells.

Alternatively, the heavy chain variable domain defined by SEQ ID NO. 49 may be paired with the light chain variable domain defined by SEQ ID NO. 50 to form an antigen binding site that can bind to NKG2D, as shown in US 9,273,136.

QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSS(SEQ ID NO:49)

QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL(SEQ ID NO:50)

Alternatively, the heavy chain variable domain defined by SEQ ID NO. 51 may be paired with the light chain variable domain defined by SEQ ID NO. 52 to form an antigen binding site that can bind to NKG2D, as shown in US 7,879,985.

QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS(SEQ ID NO:51)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK(SEQ ID NO:52)

Table 2 lists peptide sequences for the heavy and light chain variable domains, which in combination bind to CD 33.

Alternatively, a novel antigen binding site that can bind to CD33 can be determined by screening for binding to the amino acid sequence defined by SEQ ID NO 53.

SEQ ID NO:53

MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

Within the Fc domain, CD16 binding is mediated by a hinge region and a CH2 domain. For example, within human IgG1, the interaction with CD16 is mainly focused on the amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al, Nature,406(6793): 267-273). Mutations may be selected to enhance or reduce binding affinity to CD16 based on known domains, for example, by using phage display libraries or yeast surface display cDNA libraries, or mutations may be designed based on the three-dimensional structure of known interactions.

Assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which can result in assembly of homodimers as well as heterodimers for each antibody heavy chain. Promoting preferential assembly of heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US13/494870, US16/028850, US11/533709, US12/875015, US13/289934, US14/773418, US12/811207, US13/866756, US14/647480, and US 14/830336. For example, based on human IgG1, and incorporating within the first and second polypeptides different pairs of amino acid substitutions that allow the two chains to selectively heterodimerize with each other, mutations can be made in the CH3 domain. The positions of the amino acid substitutions shown below are all numbered according to the EU index in Kabat.

In one instance, the amino acid substitution in the first polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in the second polypeptide replaces the original amino acid (S) with a smaller amino acid (or smaller amino acids) selected from alanine (a), serine (S), threonine (T), or valine (V), such that the larger amino acid substitution (protuberance) fits the surface of the smaller amino acid substitution (cavity). For example, one polypeptide may comprise the substitution T366W, and another may comprise three substitutions, including T366S, L368A, and Y407V.

The antibody heavy chain variable domains of the invention may optionally be coupled to amino acid sequences at least 90% identical to an antibody constant region, such as an IgG constant region comprising hinge, CH2 and CH3 domains with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region compared to the human IgG1 constant region, e.g., at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and/or K439. Exemplary substitutions include, for example, Q347, Y349, T350, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, K439 and K439.

In certain embodiments, mutations in CH1 that may be incorporated into the constant region of human IgG1 may be located at amino acids V125, F126, P127, T135, T139, a140, F170, P171, and/or V173. In certain embodiments, mutations in ck that may be incorporated into the constant region of human IgG1 may be located at amino acids E123, F116, S176, V163, S174, and/or T164.

The amino acid substitutions may be selected from the following groups of substitutions shown in table 3.

Alternatively, the amino acid substitutions may be selected from the following groups of substitutions shown in table 4.

TABLE 4
				A first polypeptide	A second polypeptide
Group 1	K409W	D399V/F405T
			Group 2	Y349S	E357W
Group 3	K360E	Q347R
			Group 4	K360E/K409W	Q347R/D399V/F405T
Group 5	Q347E/K360E/K409W	Q347R/D399V/F405T
			Group 6	Y349S/K409W	E357W/D399V/F405T

Alternatively, the amino acid substitutions may be selected from the following groups of substitutions shown in table 5.

TABLE 5
				A first polypeptide	A second polypeptide
Group 1	T366K/L351K	L351D/L368E
			Group 2	T366K/L351K	L351D/Y349E
Group 3	T366K/L351K	L351D/Y349D
			Group 4	T366K/L351K	L351D/Y349E/L368E
Group 5	T366K/L351K	L351D/Y349D/L368E
			Group 6	E356K/D399K	K392D/K409D

Alternatively, the at least one amino acid substitution in each polypeptide chain can be selected from table 6.

Alternatively, the at least one amino acid substitution may be selected from the group of substitutions in table 7, wherein a position (or positions) indicated in the first polypeptide column is substituted with any known negatively charged amino acid, and a position (or positions) indicated in the second polypeptide column is substituted with any known positively charged amino acid.

TABLE 7
		A first polypeptide	A second polypeptide
K392, K370, K409 or K439	D399, E356 or E357

Alternatively, the at least one amino acid substitution may be selected from the group in table 8, wherein a position (or positions) indicated in the first polypeptide column is substituted with any known positively charged amino acid, and a position (or positions) indicated in the second polypeptide column is substituted with any known negatively charged amino acid.

TABLE 8
		A first polypeptide	A second polypeptide
d399, E356 or E357	K409, K439, K370 or K392

Alternatively, the at least one amino acid substitution may be selected from the following group in table 9.

TABLE 9
		A first polypeptide	A second polypeptide
T350V, L351Y, F405A and Y407V	T350V, T366L, K392L and T394W

Alternatively or additionally, the structural stability of the heteromultimeric protein may be improved by introducing S354C on either the first or second polypeptide chain and Y349C on the opposite polypeptide chain, which forms an artificial disulfide bridge at the interface of the two polypeptides.

The multispecific proteins described above can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain may be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain may be cloned into a second expression vector; a third nucleic acid sequence encoding an immunoglobulin light chain may be cloned into a third expression vector; the first, second and third expression vectors can be stably transfected together into a host cell to produce the multimeric protein.

To achieve the highest yield of multispecific protein, different ratios of the first, second and third expression vectors can be explored to determine the optimal ratio for transfection into a host cell. After transfection, individual clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or clonipix.

The clones can be cultured under conditions suitable for bioreactor scale-up and maintain expression of the multispecific protein. Multispecific proteins may be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

Characterization of multispecific proteins

In certain embodiments, a multispecific protein described herein comprising an NKG2D binding domain and a binding domain of CD33 binds to a cell expressing human NKG 2D. In certain embodiments, the multispecific protein binds to the tumor associated antigen CD33 at levels comparable to a monoclonal antibody having the same CD33 binding domain. However, the multispecific proteins described herein are more effective than the corresponding CD33 monoclonal antibodies in reducing the growth of CD 33-expressing tumors and killing CD 33-expressing cancer cells.

In certain embodiments, a multispecific protein described herein comprising a NKG2D binding domain and a binding domain of CD33 can activate primary human NK cells when cultured with tumor cells that express the antigen CD 33. NK cell activation is characterized by degranulation of CD107a and increased production of IFN γ cytokines. Furthermore, the multispecific proteins show superior activation of human NK cells in the presence of tumor cells expressing the antigen CD33 compared to monoclonal antibodies comprising the same CD33 binding domain.

In certain embodiments, the multispecific proteins described herein comprise a NKG2D binding domain and a binding domain of CD33 that enhances the activity of quiescent and IL-2 activated human NK cells in the presence of tumor cells expressing the antigen CD 33.

In certain embodiments, the multispecific proteins described herein comprise a NKG2D binding domain and a binding domain of the tumor-associated antigen CD33, enhancing the cytotoxic activity of quiescent and IL-2 activated human NK cells in the presence of tumor cells expressing the antigen CD 33. In certain embodiments, multispecific proteins may provide advantages over corresponding monoclonal antibodies against tumor cells expressing neutralizing low CD 33.

In certain embodiments, the multispecific proteins described herein may be advantageous for treating cancers with highly expressed Fc receptors (fcrs) or cancers present in a tumor microenvironment with high levels of fcrs, as compared to the corresponding CD33 monoclonal antibody. Monoclonal antibodies act on tumor growth through a variety of mechanisms, including ADCC, CDC, phagocytosis, and signal blocking, among others. Among Fc γ rs, CD16 has the lowest affinity for IgG Fc; fc γ RI (CD64) is a high affinity FcR that binds IgGFc 1000-fold stronger than CD 16. CD64 is commonly expressed in many hematopoietic lineages, such as the myeloid lineage, and can be expressed in tumors derived from these cell types, such as Acute Myeloid Leukemia (AML). Immune cells, such as MDSCs and monocytes, that infiltrate into the tumor also express CD64 and are known to infiltrate the tumor microenvironment. Expression of CD64 in the tumor or tumor microenvironment can adversely affect monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to engage CD16 on the NK cell surface, as antibodies preferentially bind to high affinity receptors. Multispecific proteins can overcome the adverse effects of CD64 expression (in the tumor or tumor microenvironment) in monoclonal antibody therapy by targeting two activating receptors on the surface of NK cells. Regardless of CD64 expression on tumor cells, the multispecific protein is able to mediate human NK cell responses against all tumor cells, as dual targeting of two activating receptors on NK cells provides for stronger specific binding to NK cells.

In some embodiments, the multispecific proteins described herein may provide better safety through reduced side effects targeting to off-tumor targets. Both natural killer cells and CD8T cells were able to directly lyse tumor cells, but NK cells and CD8T cells recognize different mechanisms from normal self cells and tumor cells. The activity of NK cells is regulated by the balance of signals from activating receptors (NCR, NKG2D, CD16, etc.) and inhibitory receptors (KIR, NKG2A, etc.). The balance of these activation and inhibition signals allows NK cells to identify healthy self-cells from among stressed, virally infected, or transformed self-cells. This "built-in" self-tolerance mechanism will help protect normal healthy tissue from NK cell responses. To extend this principle, NK cell self-tolerance would allow TriNKET to target antigens expressed on itself and on tumors without extratumoral side effects, or with an increased therapeutic window. Unlike natural killer cells, T cells need to recognize specific peptides presented by MHC molecules for activation and effector functions. T cells have become a major target for immunotherapy and a number of strategies have been developed to redirect T cell responses to tumors. T cell bispecific antibodies, checkpoint inhibitors, and CAR-T cells have all received FDA approval, but often suffer from dose-limiting toxicity. T cell bispecific antibodies and CAR-T cells function around the TCR-MHC recognition system by targeting antigens on the surface of tumor cells using a binding domain and transducing activation signals into effector cells using an engineered signaling domain. While effective in eliciting anti-tumor immune responses, these therapies are often accompanied by Cytokine Release Syndrome (CRS) and side effects that target off-tumor targets. Multispecific proteins are unique in this respect because they do not "cross" the natural system of NK cell activation and inhibition. In contrast, multispecific proteins are intended to influence the balance and provide additional activation signals to NK cells while maintaining NK tolerance to the healthy itself.

In some embodiments, the multispecific proteins described herein delay the progression of a tumor more effectively than a corresponding CD33 monoclonal antibody comprising the same CD33 binding domain. In some embodiments, the multispecific proteins described herein may be more effective against cancer metastasis than a corresponding CD33 monoclonal antibody comprising the same CD33 binding domain.

Therapeutic applications

The present invention provides methods of treating cancer using the multispecific binding proteins described herein and/or the pharmaceutical compositions described herein. The methods can be used to treat various cancers that express CD33 by administering to a patient in need thereof a therapeutically effective amount of a multispecific binding protein described herein.

The treatment method may be characterized according to the cancer to be treated. For example, in certain embodiments, the cancer is AML, myelodysplastic syndrome, chronic myelomonocytic leukemia, myeloid blast phase of chronic myelogenous leukemia, and ALL.

In certain other embodiments, the cancer is brain, breast, cervical, colon, colorectal, endometrial, esophageal, leukemia, lung, liver, melanoma, ovarian, pancreatic, rectal, kidney, stomach, testicular, or uterine cancer. In yet other embodiments, the cancer is squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid gland carcinoma, biliary tract carcinoma, thyroid carcinoma, acral freckle melanoma, actinic keratosis, acute lymphocytic leukemia, acute myelogenous leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, anal canal carcinoma, anal carcinoma, anorectal carcinoma, astrocytoma, bartholinial carcinoma, basal cell carcinoma, cholangiocarcinoma, bone carcinoma, bone marrow carcinoma, bronchial adenocarcinoma, carcinoid carcinoma, cholangiocarcinoma, chondrosarcoma, choroidal plexus papilloma/carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, digestive system carcinoma, duodenal carcinoma, endocrine system carcinoma, endoblastoma, neuroblastoma, melanoma, neuroblastoma, adenocarcinoma of the biliary tree, adenocarcinoma of the kidney, Endometrial hyperplasia, endometrial interstitial sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, ewing's sarcoma, cancer of the eye and orbit, female genital cancer, focal nodular hyperplasia, cancer of the gallbladder, cancer of the antrum, cancer of the fundus stomach, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioma, hepatoadenoma, hepatoadenomatosis, cancer of the hepatic bile duct, hepatocellular carcinoma, hodgkin's disease, ileocecum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell tumor, intrahepatic bile duct cancer, invasive squamous cell carcinoma, hollow bowel cancer, joint cancer, kaposi's sarcoma, pelvic cancer, large cell cancer, large bowel cancer, leiomyosarcoma, malignant lentiginous melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelioma, medulloblastoma, neuroblastoma, cervical cancer, medullary epithelial tumors, meninges cancer, mesothelial cancer, metastatic cancer, oral cancer, mucoepidermoid cancer, multiple myeloma, muscle cancer, nasal cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-hodgkin's lymphoma, oat cell cancer, oligodendroglial cancer, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonal blastoma, rectal cancer, renal cell cancer, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cancer, sinus cancer, skin cancer, small cell cancer, small bowel cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spinal column cancer, squamous cell cancer, striated muscle cancer, mesothelial cancer, superficial diffuse melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, cancer of the lung, cervical cancer, cervical, Urinary tract cancer, urinary bladder cancer, urinary system cancer, cervical cancer, uterine corpus cancer, uveal melanoma, vaginal cancer, wart cancer, intestinal peptide tumor of blood vessels, vulvar cancer, high differentiation cancer or Wilms' tumor.

In certain other embodiments, the cancer is a non-hodgkin's lymphoma, such as a B cell lymphoma or a T cell lymphoma. In certain embodiments, the non-hodgkin's lymphoma is a B cell lymphoma, such as diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, extranodal marginal zone B cell lymphoma, lymph node marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma, burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary Central Nervous System (CNS) lymphoma. In certain other embodiments, the non-hodgkin's lymphoma is a T cell lymphoma, such as a precursor T-lymphoblastic lymphoma, a peripheral T cell lymphoma, a cutaneous T cell lymphoma, an angioimmunoblastic T cell lymphoma, an extranodal natural killer/T cell lymphoma, an enteropathy-type T cell lymphoma, a subcutaneous panniculitis-like T cell lymphoma, an anaplastic large cell lymphoma, or a peripheral T cell lymphoma.

The cancer to be treated can be characterized by the presence of a particular antigen expressed on the surface of the cancer cell. In certain embodiments, in addition to CD33, the cancer cells may express one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD 1.

Combination therapy

Another aspect of the invention provides combination therapy. The multispecific binding proteins described herein may be used in combination with additional therapeutic agents to treat cancer.

Exemplary therapeutic agents that may be used as part of a combination therapy for the treatment of cancer include, for example, radiation, mitomycin, tretinoin, bendamustine, gemcitabine, vincristine, etoposide, cladribine, dibromomannitol, methotrexate, doxorubicin, carboquinone, pentostatin, nitrorubine (nitracrine), neat staudine, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sofalclatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, etimide, ketanserin, doxifluridine, etretinate, isotretinoin, streptozotocin, nimustine, vindesine, flutamide, butocin, doxin, furazone, furazoline, cerbrozoplatin, platinum, dulcitol, euonymus, and flutamaride, Tegafur, ifosfamide, prednisostine, saproline, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetalone, tamoxifen, progesterone, meindroxane, epithioandrostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony stimulating factor-1, colony stimulating factor-2, dinierein, interleukin-2, luteinizing hormone releasing factor, and variants of the above agents that may exhibit differential binding to their cognate receptors, as well as increased or decreased serum half-life.

Another class of agents that can be used as part of a combination therapy to treat cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of the following: (i) cytotoxic T lymphocyte-associated antigen 4(CTLA4), (ii) programmed cell death protein 1(PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM 3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. food and drug administration for the treatment of melanoma.

Still other agents that may be used as part of a combination therapy to treat cancer are monoclonal antibody agents (e.g., herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors) that target non-checkpoint targets.

Still other classes of anti-cancer agents include, for example: (i) an inhibitor selected from an ALK inhibitor, an ATR inhibitor, an A2A antagonist, a base excision repair inhibitor, a Bcr-Abl tyrosine kinase inhibitor, bruton's tyrosine kinase inhibitor, a CDC7 inhibitor, a CHK1 inhibitor, a cyclin-dependent kinase inhibitor, a DNA-PK inhibitor, an inhibitor of both DNA-PK and mTOR, a DNMT1 inhibitor, a DNMT1 inhibitor plus 2-chloro-deoxyadenosine, an HDAC inhibitor, a hedgehog signaling pathway inhibitor, an IDO inhibitor, a JAK inhibitor, an mTOR inhibitor, a MEK inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (ii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from the group consisting of IL-12, IL-15, GM-CSF and G-CSF.

The protein of the invention can also be used as an auxiliary means for surgical excision of primary lesions.

The amount and relative timing of administration of the multispecific binding protein and the additional therapeutic agent may be selected so as to achieve the desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need of such administration, the therapeutic agents in the combination or the pharmaceutical composition or compositions comprising the therapeutic agents can be administered in any order, e.g., sequentially, co-administered, administered together, administered simultaneously, etc. Further, for example, the multispecific binding protein may be administered during the time when the additional therapeutic agent(s) exert their prophylactic or therapeutic effect or vice versa.

V. pharmaceutical composition

The disclosure also features pharmaceutical compositions comprising a therapeutically effective amount of a protein described herein. The compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for suitable formulation. Suitable formulations for use in the present disclosure are found in Remington's pharmaceutical sciences, Mack Publishing Company, philiadelphia, Pa., 17 th edition, 1985. For a brief review of drug delivery methods, see, e.g., Langer (Science 249: 1527) -1533, 1990).

The intravenous drug delivery formulation of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to a channel containing a tube and/or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 tablets. In certain embodiments, the formulation may be lyophilized, and 45mg of the lyophilized formulation may be contained in one vial. In certain embodiments, about 40mg to about 100mg of the lyophilized formulation may be contained in one vial. In certain embodiments, lyophilized formulations from 12, 27 or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous pharmaceutical formulation. In certain embodiments, the formulation may be a liquid formulation and stored at about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 250 mg/vial.

The present disclosure may be present in a liquid aqueous pharmaceutical formulation comprising a therapeutically effective amount of a protein in a buffered solution forming the formulation.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized formulation being combined with a sterile aqueous carrier prior to administration. The pH of the formulation is typically from 3 to 11, more preferably from 5 to 9 or from 6 to 8, most preferably from 7 to 8, such as from 7 to 7.5. The resulting composition in solid form may be packaged in a plurality of single dosage units, each dosage unit containing a fixed amount of the above-mentioned agent or agents. The composition in solid form may also be packaged in containers to obtain flexible amounts.

In certain embodiments, the present disclosure provides formulations with extended shelf life comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

In certain embodiments, an aqueous formulation is prepared comprising a protein of the present disclosure in a pH buffered solution. The buffer of the invention may have a pH of about 4 to about 8, for example about 4.5 to about 6.0 or about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. The intermediate ranges of pH mentioned above are also intended to be part of the present disclosure. For example, ranges of values using any combination of the above values as upper and/or lower limits are intended to be included. Examples of buffers to control the pH within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, histidines, citrates, and other organic acid buffers.

In certain embodiments, the formulation comprises a buffer system comprising citrate and phosphate to maintain the pH in the range of about 4 to about 8. In certain embodiments, the pH range may be a pH range of about 4.5 to about 6.0, or about pH4.8 to about 5.5, or about 5.0 to about 5.2. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3mg/ml citric acid (e.g., 1.305mg/ml), about 0.3mg/ml sodium citrate (e.g., 0.305mg/ml), about 1.5mg/ml disodium phosphate dihydrate (e.g., 1.53mg/ml), about 0.9mg/ml sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2mg/ml sodium chloride (e.g., 6.165 mg/ml). In certain embodiments, the buffer system comprises 1-1.5mg/ml citric acid, 0.25 to 0.5mg/ml sodium citrate, 1.25 to 1.75mg/ml disodium phosphate dihydrate, 0.7 to 1.1mg/ml sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4mg/ml sodium chloride. In certain embodiments, sodium hydroxide is used to adjust the pH of the formulation.

Polyols, which act as tonicity agents and can stabilize antibodies, may also be included in the formulation. The polyols are added to the formulation in amounts that may vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also vary depending on the molecular weight of the polyol. For example, a smaller amount of monosaccharide (e.g., mannitol) may be added than a disaccharide (e.g., trehalose). In certain embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg/ml. In certain embodiments, the concentration of mannitol may be about 7.5 to 15 mg/ml. In certain embodiments, the concentration of mannitol may be about 10-14 mg/ml. In certain embodiments, the concentration of mannitol may be about 12 mg/ml. In certain embodiments, the polyol sorbitol may be included in the formulation.

Detergents or surfactants may also be added to the formulation. Exemplary detergents include non-ionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of particulates in the formulation and/or reduces adsorption. In certain embodiments, the formulation may comprise a surfactant that is a polysorbate. In certain embodiments, the formulation may comprise the detergent polysorbate 80 or tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon derHifsstoffe, Editiocantor Verlag Aulendorf, 4 th edition, 1996). In certain embodiments, the formulation may comprise from about 0.1mg/mL to about 10mg/mL of polysorbate 80 or from about 0.5mg/mL to about 5mg/mL of polysorbate 80. In certain embodiments, the formulation may add about 0.1% polysorbate 80.

In embodiments, the protein products of the present disclosure are formulated as liquid formulations. The liquid formulation can be provided at a concentration of 10mg/mL in a USP/Ph Eur type I50R vial closed with a rubber stopper and sealed with an aluminum crimp closure. The stopper may be made of an elastomer conforming to USP and Ph Eur. In certain embodiments, the vial may be filled with 61.2mL of the protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with a 0.9% saline solution.

In certain embodiments, the liquid formulations of the present disclosure may be prepared as a solution at a concentration of 10mg/mL in combination with a steady level of sugar. In certain embodiments, the liquid formulation may be prepared in an aqueous carrier. In certain embodiments, the amount of stabilizer added may be no greater than a viscosity that may result in undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may further comprise one or more of a buffer, a surfactant, and a preservative.

In certain embodiments, the pH of the liquid formulation may be set by the addition of a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.

In addition to polymerization, deamidation is a common product variant of peptides and proteins, which can occur during fermentation, harvesting/cell clarification, purification, drug/drug storage, and during sample analysis. Deamidation is the NH in a protein that forms a succinimide intermediate that can undergo hydrolysis₃Is lost. The succinimide intermediate results in a 17 dalton mass reduction of the parent peptide. Subsequent hydrolysis resulted in an 18 dalton mass increase. Due to instability under aqueous conditions, it is difficult to isolate the succinimide intermediate. Thus, deamidation is typically detectable as a1 dalton mass increase. Deamidation of asparagine produces aspartic acid or isoaspartic acid. Parameters that affect the deamidation rate include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser after Asn in the protein sequence lead to higher sensitivity to deamidation.

In certain embodiments, the liquid formulations of the present disclosure may be stored under pH and humidity conditions to prevent deamination of the protein product.

The aqueous carrier of interest herein is a pharmaceutically acceptable aqueous carrier (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-purpose (multi-dose) formulation.

Intravenous (IV) formulations may be the preferred route of administration in certain circumstances, for example when patients receive all drugs via the IV route in hospitals after transplantation. In certain embodiments, the liquid formulation is diluted with a 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

In certain embodiments, the salt or buffer component may be added in an amount of 10mM to 200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) having "base forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may serve as the counter ion.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-use (multi-dose) formulation.

The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, ringer's solution, or dextrose solution.

The present disclosure may reside in a lyophilized formulation comprising a protein and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may further comprise one or more of a buffer, a surfactant, a bulking agent, and/or a preservative.

The amount of sucrose or maltose that can be used to stabilize the lyophilized drug product can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1: 5.

In certain embodiments, the pH of the formulation may be set by the addition of a pharmaceutically acceptable acid and/or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

Prior to lyophilization, the pH of a solution comprising a protein of the present disclosure may be adjusted to 6 to 8. In certain embodiments, the pH of the lyophilized drug product may range from 7 to 8.

In certain embodiments, the salt or buffer component may be added in an amount of 10mM to 200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) having "base forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may serve as the counter ion.

In certain embodiments, a "filler" may be added. A "bulking agent" is a compound that can increase the quality of the lyophilized mixture and aid in the physical structure of the lyophilized cake (e.g., aid in preparing a substantially uniform lyophilized cake that retains an open cell structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulation of the present invention may contain such a bulking agent.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the preparation of a multi-use (multi-dose) formulation.

In certain embodiments, the lyophilized pharmaceutical product can be formulated with an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and can be used to prepare a liquid formulation after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, ringer's solution, or dextrose solution.

In certain embodiments, the lyophilized pharmaceutical product of the present disclosure is reconstituted with sterile water for injection USP (swfi) or 0.9% sodium chloride injection USP. During reconstitution, the lyophilized powder dissolves into solution.

In certain embodiments, the lyophilized protein product of the present disclosure is formulated into about 4.5mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The specific dose may be a uniform dose, for example, 50-5000mg of protein, for each patient. Alternatively, the dosage for the patient may be tailored to the approximate weight or surface area of the patient. Other factors in determining an appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration and the age, sex and physical condition of the patient. The calculations necessary to determine an appropriate therapeutic dose are routinely further refined by those skilled in the art, particularly in light of the dose information and analysis disclosed herein. Dosages can also be determined by using known assays for determining the dosage to be used in conjunction with appropriate dose-response data. The dosage of an individual patient may be adjusted based on monitoring of disease progression. The blood level of the targetable construct or complex in the patient can be measured to see if the dose needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and/or complexes and their doses are most likely to be effective in a given individual (Schmitz et al, Clinica Chimica Acta 308:43-53,2001; Steimer et al, Clinica Chimica Acta 308:33-41,2001).

Typically, the dosage on a weight basis is from about 0.01 μ g to about 100mg/kg body weight, such as from about 0.01 μ g to about 100mg/kg body weight, from about 0.01 μ g to about 50mg/kg body weight, from about 0.01 μ g to about 10mg/kg body weight, from about 0.01 μ g to about 1mg/kg body weight, from about 0.01 μ g to about 100 μ g/kg body weight, from about 0.01 μ g to about 50 μ g/kg body weight, from about 0.01 μ g to about 10 μ g/kg body weight, from about 0.01 μ g to about 1 μ g/kg body weight, from about 0.01 μ g to about 0.1 μ g/kg body weight, from about 0.1 μ g to about 100mg/kg body weight, from about 0.1 μ g to about 50mg/kg body weight, from about 0.1 μ g to about 10mg/kg body weight, from about 0.1 μ g to about 1mg/kg body weight, from about 0.1 μ g to about 100 μ g/kg body weight, from about 0.1 μ g to about 10 μ g/kg body weight, About 0.1 μ g to about 1 μ g/kg body weight, about 1 μ g to about 100mg/kg body weight, about 1 μ g to about 50mg/kg body weight, about 1 μ g to about 10mg/kg body weight, about 1 μ g to about 1mg/kg body weight, about 1 μ g to about 100 μ g/kg body weight, about 1 μ g to about 50 μ g/kg body weight, about 1 μ g to about 10 μ g/kg body weight, about 10 μ g to about 100mg/kg body weight, about 10 μ g to about 50mg/kg body weight, about 10 μ g to about 10mg/kg body weight, about 10 μ g to about 1mg/kg body weight, about 10 μ g to about 100 μ g/kg body weight, about 10 μ g to about 50 μ g/kg body weight, about 50 μ g to about 100mg/kg body weight, about 50 μ g to about 50mg/kg body weight, about 50 μ g to about 10mg/kg body weight, About 50 μ g to about 1mg/kg body weight, about 50 μ g to about 100 μ g/kg body weight, about 100 μ g to about 100mg/kg body weight, about 100 μ g to about 50mg/kg body weight, about 100 μ g to about 10mg/kg body weight, about 100 μ g to about 1mg/kg body weight, about 1mg to about 100mg/kg body weight, about 1mg to about 50mg/kg body weight, about 1mg to about 10mg/kg body weight, about 10mg to about 100mg/kg body weight, about 10mg to about 50mg/kg body weight, about 50mg to about 100mg/kg body weight.

The dose may be administered one or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. One of ordinary skill in the art can readily estimate the repetition rate of administration based on the measured residence time and the concentration of targetable construct or complex in the body fluid or tissue. Administration of the invention may be intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intrapleural administration, intrathecal administration, intracavity administration, by catheter infusion or by direct intralesional injection. This may be administered one or more times per day, one or more times per week, one or more times per month, and one or more times per year.

The above description describes various aspects and embodiments of the present invention. The patent application specifically contemplates all combinations and permutations of aspects and embodiments.

Examples

The invention now generally described will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the invention, and are not intended to limit the invention.