EA043960B1 - ANTIGEN-BINDING SITES THAT BIND TO THE NATURAL KILLER GROUP 2D RECEPTOR - Google Patents

Description

Cross references to related applications

This application claims the benefit and priority of U.S. Provisional Patent Application No. 62/628,161, filed February 8, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes; and US Provisional Patent Application No. 62/716259, filed August 8, 2018.

Field of invention

The present invention provides proteins with antibody heavy chain and light chain variable domains that can be paired to form an antigen binding site targeting natural killer cell group 2D (NKG2D) receptor on the surface of natural killer cells, pharmaceutical compositions containing such proteins, and therapeutic methods with the use of such proteins and pharmaceutical compositions, including for the treatment of cancer.

State of the art

Cancer continues to be a major health problem despite significant research efforts and scientific advances reported in the treatment literature for this disease. Some of the most commonly diagnosed cancers include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of cancer in men. Breast cancer remains the leading cause of death in women. Current treatment options for these cancers are not effective for all patients and/or may have significant side effects. Other types of cancer also remain difficult to treat with current treatment options.

Anticancer immunotherapy agents are desirable because of their high specificity and ability to promote the destruction of cancer cells using the patient's own immune system. Fusion proteins, such as bispecific T cell recruiters, are anticancer immunotherapy agents described in the literature that bind to tumor cells and T cells to facilitate tumor cell destruction. Antibodies that bind to certain tumor-associated antigens and to certain immune cells are 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 make up approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for priming, which distinguishes them from T cells. Activated NK cells kill target cells in a similar manner to cytotoxic T cells, i.e. through cytolytic granules that contain perforin and granzymes, and through death receptor pathways. Activated NK cells also secrete inflammatory cytokines, such as IFN-gamma and chemokines, which promote the recruitment of other leukocytes to target tissues.

NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIR). Alternatively, when NK cells encounter cancer cells, they are activated through their activating receptors (eg, NKG2D, NCRs, DNAM1). NK cells are also activated by the constant region of some immunoglobulins through CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein that is expressed by essentially all natural killer cells, where NKG2D serves as an activating receptor. The ability to modulate NK cell function by NKG2D is useful in various therapeutic contexts, including cancer.

The essence of the invention

Antibodies to NKG2D have been identified that provide important advantages in the development of therapeutic agents. For example, some of these antibodies do not simply bind the human NKG2D receptor, but have one or more additional benefits, such as the ability to agonize the receptor; the ability to compete with a natural ligand for receptor binding and/or the ability to cross-react with NKG2D from other species such as the cynomolgus macaque. These benefits may be achieved through a range of affinities for NKG2D.

Accordingly, one aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGS TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAPNYGDTTHDYYYMD VWGKGTTVTVSS (SEQ Ш NO:1, ADI-29379).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 1. In some embodiments, the heavy chain variable domain includes the amino acid sequences YTFTSYYMH (SEQ ID NO: 11) as the first complementarity determining region 1 (CDR1 ), IINPSGGSTSYAQKFQG (SEQ ID NO: 12) as the second CDR (CDR2) and ARGAPNYGDTTHDYYYMDV (SEQ ID NO: 13) as

- 1 043960 as the third CDR (CDR3) SEQ ID NO: 1. In some embodiments, the heavy chain variable domain includes the amino acid sequences SYYMH (SEQ ID NO: 45) as

CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO: 12) as CDR2 and GAPNYGDTTHDYYYMDV (SEQ ID NO: 68) as CDR3 SEQ ID NO: 1.

Another aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSG GTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDV WGQGTTVTVSS (SEQ ID NO: 3, ADI-29463).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 3. In some embodiments, the heavy chain variable domain includes the amino acid sequences YTFTGYYMH (SEQ ID NO: 17) as the first complementarity determining region (CDR1) , WINPNSGGTNYAQKFQG (SEQ ID NO: 18) as the second CDR (CDR2) and ARDTGEYYDTDDHGMDV (SEQ ID NO: 19) as the third CDR (CDR3) SEQ ID NO: 3. In some embodiments, the heavy chain variable domain includes the amino acid sequences GYYMH (SEQ ID NO: 92) as CDR1, WINPNSGGTNYAQKFQG (SEQ ID NO: 18) as CDR2, and DTGEYYDTDDHGMDV (SEQ ID NO: 69) as CDR3 SEQ ID NO: 3.

Another aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTL VTVSS (SEQ Ш NO:5, ADI-27744).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 5. In some embodiments, the heavy chain variable domain includes the amino acid sequences FTFSSYAMS (SEQ ID NO: 23) as the first complementarity determining region (CDR1) , AISGSGGSTYYADSVKG (SEQ ID NO: 24) as the second CDR (CDR2) and AKDGGYYDSGAGDY (SEQ ID NO: 25) as the third CDR (CDR3) SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises SYAMS amino acid sequences (SEQ ID NO: 47) as CDR1, AISGSGGSTYYADSVKG (SEQ ID NO: 24) as CDR2, and DGGYYDSGAGDY (SEQ ID NO: 70) as CDR3 SEQ ID NO: 5.

Another aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity:

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQG TLVTVSS (SEQ ID NO:7, ADI-27749).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain includes the amino acid sequences FTFSSYSMN (SEQ ID NO: 29) as the first complementarity determining region (CDR1) , SISSSSSYIYYADSVKG (SEQ ID NO: 30) as the second CDR (CDR2) and ARGAPMGAAAGWFDP (SEQ ID NO: 31) as the third CDR (CDR3) SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYSMN (SEQ ID NO: 48) as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO: 30) as CDR2, and GAPMGAAAGWFDP (SEQ ID NO: 71) as CDR3 SEQ ID NO: 7.

Another aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity

EVQLVESGGGLVKPGGSLRLSC AASGFTF SS YSMNWVRQAPGKGLEWVS SIS S S S SYIY YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGQGT LVTVSS (SEQ ID NO:85, A49MI).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 85. In some embodiments, the heavy chain variable domain includes the amino acid sequences FTFSSYSMN (SEQ ID NO: 29) as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO : 30) as CDR2 and ARGAPIGAAAGWFDP (SEQ ID NO: 77) as CDR3 SEQ ID NO: 85. In some embodiments, the heavy chain variable domain includes the amino acid sequences SYSMN (SEQ ID NO: 48) as CDR1, SISSSSSYIYYADSVKG (SEQ ID NO: 30) as CDR2 and GAPIGAAAGWFDP (SEQ ID NO: 78) as CDR3 SEQ ID NO: 85.

Another aspect of the present invention relates to an antibody heavy chain variable domain having at least 90% amino acid sequence identity

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QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGS TSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGAGFAYGMDYYYMDV WGKGTTVTVSS (SEQ ID NO:9, ADI-29378).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 9. In some embodiments, the heavy chain variable domain includes the amino acid sequences YTFTSYYMH (SEQ ID NO: 35) as the first complementarity determining region (CDR1) , IINPSGGSTSYAQKFQG (SEQ ID NO: 36) as the second CDR (CDR2) and AREGAGFAYGMDYYYMDV (SEQ ID NO: 37) as the third CDR (CDR3) SEQ ID NO: 9. In some embodiments, the heavy chain variable domain includes the amino acid sequences SYYMH (SEQ ID NO: 45) as CDR1, IINPSGGSTSYAQKFQG (SEQ ID NO: 36) as CDR2, and EGAGFAYGMDYYYMDV (SEQ ID NO: 72) as CDR3 SEQ ID NO: 9.

The heavy chain variable domain of an antibody of the present invention may optionally be combined with an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region, including 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, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to the constant region of an antibody from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be included in the constant region compared to the human IgG1 constant region, for example in 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. Typical replacements include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K36OE, K360W, Q3 62E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K3 92E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

In some embodiments, mutations that may be included in the CH1 constant region of human IgG1 may be at amino acid V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In some embodiments, mutations that may be included in the human IgG1 constant region CK may be at amino acid E123, F116, S176, V163, S174, and/or T164.

In some embodiments, one of the heavy chain variable domains described herein is combined with a light chain variable domain to form an antigen binding region capable of binding NKG2D. For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 may be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence

EIVMTQ SPATES VSPGERATLSCRASQ SVS SNLAWYQQKPGQAPRLLIYGASTRATGIP A RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDDWPFTFGGGTKVEIK (SEQ ID NO:2, ADI29379).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 2. In some embodiments, the heavy chain variable domain includes the amino acid sequences RASQSVSSNLA (SEQ ID NO: 14) as the first complementarity determining region (CDR) , GASTRAT (SEQ ID NO: 15) as the second CDR and QQYDDWPFT (SEQ ID NO: 16) as the third CDR.

For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3 may be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence

EIVLTQSPGTLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPAR FSGSGSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK (SEQ ID NO:4, ADI29463).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 4. In some embodiments, the heavy chain variable domain includes the amino acid sequences RASQSVSSNLA (SEQ ID NO: 20) as the first complementarity determining region (CDR) , GASTRAT (SEQ ID NO: 21) as the second CDR and QQDDYWPPT (SEQ ID NO: 22) as the third CDR.

For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5 may be paired with a light chain variable domain.

- 3 043960 antibody chains with at least 90% identical amino acid sequence

DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK (SEQ ID NO:6, ADI27744).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 6. In some embodiments, the heavy chain variable domain includes the amino acid sequences RASQGIDSWLA (SEQ ID NO: 26) as the first complementarity determining region (CDR) , AASSLQS (SEQ ID NO: 27) as the second CDR and QQGVSYPRT (SEQ ID NO: 28) as the third CDR.

For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7 or 85 may be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK (SEQ ID NO:8, ADI27749).

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 8. In some embodiments, the heavy chain variable domain includes the amino acid sequences RASQGISSWLA (SEQ ID NO: 32) as the first complementarity determining region (CDR) , AASSLQS (SEQ ID NO: 33) as the second CDR and QQGVSFPRT (SEQ ID NO: 34) as the third CDR.

For example, an antibody heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 may be paired with an antibody light chain variable domain that is at least 90% identical to the amino acid sequence

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSDNWPFTFGGGTKVEIK (SEQ ID NO: 10, ADI-29378).

In some embodiments, the light chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 10. In some embodiments, the heavy chain variable domain includes the amino acid sequences RASQSVSSYLA (SEQ ID NO: 38) as the first complementarity determining region (CDR) , DASNRAT (SEQ ID NO: 39) as the second CDR and QQSDNWPFT (SEQ ID NO: 40) as the third CDR.

When a heavy chain variable domain is combined with a light chain variable domain to form an antigen binding region capable of binding NKG2D, the antigen binding region can be included in various structures, for example in a typical antibody structure with two identical heavy chains and two identical light chains forming a pair of antigen binding regions, capable of binding NKG2D; bispecific, trispecific, tetraspecific or other multispecific antibody; or a smaller structure such as scFv (in which a heavy chain variable domain is linked to a light chain variable domain).

In some embodiments, any NKG2D antigen-binding region disclosed in the present invention is included in a protein that also includes a separate antigen-binding region that binds to a tumor-associated antigen, which may allow the protein to simultaneously interact with an NK cell and a tumor cell. The tumor-associated antigen, for example, may be CD33, HER2, EpCAM, CD2, CD3, CD8, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD37, CD38, CD40, CD45RO, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CSAp, CA-125, TAG-72, EFGR/ERBB1, IGF1R, HER2, HER3, HER4, IGF-1R, c- Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA and IL- 6, MAGE-A3, B7.1, B7.2, CTLA4 or PD1.

In some embodiments, any NKG2D binding site disclosed in the present invention is included in a protein that also contains a tumor-associated antigen region and a CD16 binding site. The CD16 binding site may be an additional antigen binding region or an antibody constant region or a portion thereof, such as an IgG1 constant region (which may optionally include one or more mutations affecting, for example, effector activity or CD16 binding affinity).

In another aspect of the present invention, a method is provided for enhancing tumor cell death and treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of a protein described herein for the treatment of cancer.

Brief description of graphic materials

In fig. 1 shows a multispecific binding protein containing an NKG2D binding domain (right arm), a tumor-associated antigen binding domain (left arm), and an Fc domain or portion thereof that binds CD16.

In fig. 2 shows a multispecific binding protein containing

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An NKG2D-binding domain, a tumor-associated antigen-binding domain, any of which may be in scFv format, and an Fc domain or portion thereof that binds CD16.

In fig. 3A-3E show the NKG2D binding affinity profiles of the NKG2D binding domains measured by surface plasmon resonance.

In fig. 3A shows the NKG2D binding affinity of the NKG2D binding domain of ADI-27744 measured by surface plasmon resonance.

In fig. 3B shows the NKG2D binding affinity of the NKG2D binding domain of ADI-29379 measured by surface plasmon resonance.

In fig. 3C shows the NKG2D binding affinity of the NKG2D binding domain of ADI-27749 measured by surface plasmon resonance.

In fig. 3D shows the NKG2D binding affinity of the NKG2D binding domain of ADI-29463 measured by surface plasmon resonance.

In fig. 3E shows the NKG2D binding affinity of the NKG2D binding domain of ADI-29378 measured by surface plasmon resonance.

In fig. 4A-4H show the NKG2D competitive binding profiles of the NKG2D binding domain of ADI-27744 (A44) and ULBP6, or other anti-NKG2D antibodies, measured by surface plasmon resonance.

In fig. 4A shows the profile of an anti-NKG2D monoclonal antibody containing ADI-27744 injected over immobilized NKG2D followed by injection of ULBP6.

In fig. 4B shows the profile of ULBP6 injected on top of immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27744.

In fig. 4C shows the profile of an anti-MS monoclonal antibody injected over immobilized NKG2D, followed by injection of ULBP6.

In fig. 4D shows the profile of MS injected over immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27744.

In fig. 4E shows the profile of 1D11 injected over immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27744.

In fig. 4F shows the profile of MAB139 injected over immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27744.

In fig. 4G shows the profile of an anti-NKG2D monoclonal antibody containing ADI-27744 injected over immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27749 (A49).

In fig. 4H shows the profile of an anti-NKG2D monoclonal antibody containing ADI-27744 injected over immobilized NKG2D, followed by injection of an anti-NKG2D monoclonal antibody containing F47.

In fig. 5 are line graphs showing the binding profile of CD33-targeted TriNKET to NKG2D expressed on the surface of EL4 cells.

In fig. 6 are line graphs showing the binding profile of HER2-targeted TriNKET to NKG2D expressed on the surface of EL4 cells.

In fig. 7 are histograms showing the binding profile of CD33-targeted TriNKETs to CD33 expressed on the surface of Mv4-11 cells.

In fig. 8 are histograms showing the binding profile of HER2-targeted TriNKET to HER2 expressed on the surface of 786-O cells.

In fig. 9 are histograms showing the binding profile of HER2-targeted TriNKET to HER2 expressed on the surface of NCI-H661 cells.

In fig. 10 presents histograms showing that TriNKETs targeting HER2 mediate activation of human NK cells co-cultured with HER2-expressing NCI-H661 cells.

In fig. 11 are histograms showing that TriNKETs targeting HER2 mediate activation of human NK cells co-cultured with HER2-expressing SkBr-3 cells.

In fig. 12 are histograms showing that TriNKETs targeting CD33 mediate activation of human NK cells co-cultured with CD33-expressing human AML Mv4-11 cells.

In fig. 13 are line graphs showing that CD33-targeted TriNKETs mediate resting NK cell cytotoxicity against CD33-expressing Molm-13 cancer cells.

In fig. 14 are line graphs showing that CD33-targeted TriNKETs mediate cytotoxicity of activated NK cells against CD33-expressing Molm-13 cancer cells.

In fig. 15 shows histograms showing that HER2-targeted TriNKETs mediate cytotoxicity of resting NK cells against HER2-expressing cancer cells.

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786-O.

In fig. 16 are histograms showing that HER2-targeted TriNKETs mediate cytotoxicity of activated NK cells against HER2-expressing 786-O cancer cells.

In fig. 17 presents TriNKET in the form of triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half-antibodies, each with one light and one heavy chain, which are derived from two parent antibodies. The triomab form is a heterodimeric construct containing ½ rat antibody and ½ mouse antibody.

In fig. 18 presents TriNKET in a common light chain (LC) form that utilizes knob-in-valley (KIH) technology. KIH is a heterodimer containing two target-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations. TriNKET in KIH format can be a heterodimeric construct with two Fabs binding to target 1 and target 2 containing two different heavy chains and a common light chain that pairs with both NSs.

In fig. 19 presents TriNKET in the form of dual variable domain immunoglobulin (DVD-Ig™), which combines the target binding domains of two monoclonal antibodies through flexible natural linkers and results in a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the variable domain-targeting antigen 2 is fused to the N-terminus of the Fab-targeting antigen 1 variable domain. The design contains normal Fc.

In fig. 20 shows TriNKET in the form of an orthogonal surface Fab (Ortho-Fab), which is a heterodimeric construct that contains two Fab fragments binding to target 1 and target 2 fused to an Fc. LC-NS pairing is mediated by an orthogonal surface. Heterodimerization is mediated by mutations in Fc.

In fig. 21 presents TriNKET in Ig 2-in-1 format.

In fig. 22 shows TriNKET in ES format, which is a heterodimeric construct containing two different Fab fragments binding to target 1 and target 2 fused to Fc. Heterodimerization is mediated by electrostatic mutation in Fc.

In fig. 23 represents TriNKET in the form of Fab exchange: antibodies that exchange Fab fragments by replacing the heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies. The Fab fragment exchange form (cFae) is a heterodimer containing two target-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations.

In fig. 24 shows TriNKET in the form of a SEED body, which is a heterodimer containing two target-binding Fabs 1 and 2 and Fcs stabilized by heterodimerization mutations.

In fig. 25 presents TriNKET in the form of LuZ-Y, in which a leucine zipper is used to induce heterodimerization of two different NSs. The LuZ-Y form is a heterodimer containing two different target-binding scFabs 1 and 2 fused to an Fc. Heterodimerization is mediated by leucine zipper motifs fused to the C terminus of the Fc.

In fig. 26 presents TriNKET in the form of a Cov-X body.

In fig. 27A, 27B represent TriNKET in κλ body forms, which are heterodimeric constructs with two different Fabs fused to Fc, stabilized by heterodimerization mutations: Fab1-targeting antigen 1 contains kappa LC, while the second Fab-targeting antigen 2 contains lambda LC .

In fig. 27A is a typical representation of one κλ body shape.

In fig. 27B shows a typical representation of another κλ body.

In fig. 28 shows a heterodimeric Oasc-Fab construct that includes Fab binding to target 1 and scFab binding to target 2 fused to an Fc. Heterodimerization is mediated by mutations in Fc.

In fig. 29 shows DuetMab, which is a heterodimeric construct containing two different Antigen 1 and 2 binding Fabs and Fcs stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure proper pairing of LC and NS.

In fig. 30 shows CrossmAb, which is a heterodimeric construct with two different target-binding Fabs 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are swapped, eg CH1 is connected to VL while CL is connected to VH.

In fig. 31 shows Fit-Ig, which is a homodimeric construct where the binding of a Fab to antigen 2 is fused to the N-terminus of an HC Fab that binds to antigen 1. The construct contains a wild type Fc.

In fig. 32 is a series of line graphs demonstrating the binding of TriNKET A* and

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TriNKET A with human NKG2D according to SPR. The top panels represent kinetic fit and the bottom panels correspond to steady-state affinity.

In fig. 33 is a line graph demonstrating the effectiveness of TriNKET A and

TriNKET A* in mediating NK cell cytotoxicity towards target cells.

In fig. 34 is a line graph demonstrating the effectiveness of TriNKET A and TriNKET A* in mediating NK cell cytotoxicity to target cells.

Detailed description of the invention

The present invention provides antibody heavy chain variable domains that can be paired with antibody light chain variable domains to form an antigen binding site targeting the NKG2D receptor on natural killer cells, proteins that include NKG2D antigen binding sites, pharmaceutical compositions containing such proteins, and therapeutic methods using such proteins and pharmaceutical compositions for the treatment of cancer. Various aspects of the present invention are set forth below in the sections; however, the aspects of the present invention described in one specific section are not intended to be limited to any particular section.

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

Terms in the singular as used herein mean one or more and include the plural unless inconsistent with the context.

The terms subject and patient as used herein refer to the organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (eg, mice, monkeys, horses, cattle, pigs, dogs, cats, and the like) and more preferably include humans.

The term antigen-binding site as used herein refers to a portion of an immunoglobulin molecule that is involved in binding an antigen. In human antibodies, the antigen-binding region is formed by amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains. The three highly divergent regions in the V regions of the heavy and light chains are called hypervariable regions, which are located between more conserved flanking regions known as framework regions or FRs. Thus, the term FR refers to amino acid sequences that naturally occur between and near the hypervariable regions in immunoglobulins. In a human antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are positioned 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 called complementarity determining regions or CDRs. In some animals, such as camelids and cartilaginous fishes, the antigen-binding site is formed by a single-chain antibody forming a single-domain antibody. Antigen binding sites may exist 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 a scFv, using a peptide linker to join a heavy chain variable domain to a light chain variable domain in a single polypeptide.

The term effective amount in the context of the present invention refers to an amount of a compound (eg, a compound of the present invention) sufficient to achieve beneficial or desired results. An effective amount may be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration.

The term treatment in the context of the present invention includes any effect, such as attenuation, reduction, modulation, improvement or elimination, which results in the improvement of a pathological condition, disease, disorder, etc., or the alleviation of a symptom thereof.

The term pharmaceutical composition in the context of the present invention refers to the combination of an active agent with a carrier, inert or active, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

The term pharmaceutically acceptable carrier as used herein refers to any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions (eg, such as oil/water or water/oil emulsions) and various types of wetting agents. The compositions may also include stabilizers and preservatives. For example, carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, ^15th Ed., Mack Publ. Co., Easton, PA [1975].

Throughout the specification, where compositions are described as having, including, or containing specific components, or where processes and methods are described as having, including, or containing specific steps, it is intended that, in addition, there are compositions of the present invention that consist substantially of the listed components or consist of them, and that there are processes and methods in accordance with the present invention that consist essentially of or consist of the listed processing steps.

Generally, for compositions where a percentage is indicated, the percentage is a percentage by weight unless otherwise stated. In addition, if a variable is not accompanied by a definition, then the previous definition of the variable is taken into account.

NKG2D antigen binding site.

The present invention provides antigen binding regions that bind NKG2D and antigen heavy chain variable domains that can be used to create such antigen binding regions.

The antibody heavy chain variable domains and the light chain variable domains that they pair to form antigen binding sites capable of binding and agonizing the NKG2D receptor have now been identified and are presented in Table 1. 1. Unless otherwise indicated, the CDR sequences presented in table. 1, determined according to Kabat.

Table 1

Clones Amino acid sequence of the heavy chain variable region Amino acid lung variable region sequence chains ADI-29463 (F63) ADI-27744 (A44) QVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYMHWVRQAPGQGLEWMGII NPSGGSTSYAQKFQGRVTMTRDTSTST VYMELSSLRSEDTAVYYCARGAPNYG DTTHD YYYMD VWGKGTT VT VS S (SEQ IDNO:1) CDR1 not by Kabat (SEQ ID NO: 11) YTFTSYYMH or CDR1 (SEQ ID NO:45) - SYYMH CDR2 (SEQ ID NO :12)IWSGGSTSYAQKFQG CDR3 not by Kabat (SEQ IDNO:13)ARGAPNYGDTTHDYYYMDV or CDR3 (SEQ ID NO :68)- GAPNYGDTTHDYYYMDV QVQLVQSGAEVKKPGASVKVSCKASG YTFTGYYMHWVRQAPGQGLEWMGW INPNSGGTNYAQKFQGRVTMTRDTSIS TAYMELSRLRSDDTAVYYCARDTGEY YDTDDHGMD VWGQGTT VT VS S (SEQ ID NOG) CDR1 not by Kabat (SEQ ID NO: 17) YTFTGYYMH or CDR1 (SEQ ID NO:46) - GYYMH CDR2 (SEQ ID NO:18)- WINPNSGGTNYAQKFQG CDR3 not by Kabat (SEQ ID NO: 19) - ARDTGEYYDTDDHGMDV or CDR3 (SEQ ID NO:69)DTGEYYDTDDHGMDV EVQLLESGGGLVQPGGSLRLSCAASGF TFSSYAMSWVRQAPGKGLEWVSAISG SGGSTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKDGGYYDS GAGD YWGQGTL VT VS S (SEQ ID NO:5) CDR1 not by Kabat (SEQ ID NO:23) FTFSSYAMS or CDR1 (SEQ ID NO:47) - SYAMS EIVMTQSPATLSVSPGERATL SCRASQSVSSNLAWYQQKPG QAPRLLIYGASTRATGIPARF SSGSGSGTEFTLTISSLQSEDFA VYYCQQYDDWPFTFGGGTK VEIK (SEQIDNO:2) CDR1 (SEQ ID NO: 14)RASQSVSSNLA CDR2 (SEQ ID NO:15) GASTRAT CDR3 (SEQ ID NO:16) QQYDDWPFT EIVLTQSPGTLLSLSPGERATLS CRASQSVSSNLAWYQQKPGQ APRLLIYGASTRATGIP ARF S GSGSGTEFTLTISSLQSEDFAV YYCQQDDYWPPTFGGGTKV EIK (SEQIDNO:4) CDR1 (SEQ ID NO:20) RASQSVSSNLA CDR2 (SEQ ID NO:21) GASTRAT CDR3 (SEQ ID NO:22) QQDDYWPPT DIQMTQ SP S S VS AS VGDRVTI TCRASQGIDSWLAWYQQKP GKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDF ATYYCQQGVSYPRTFGGGTK VEIK (SEQ ID NO :6) CDR1 (SEQ ID NO:26) RASQGIDSWLA

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CDR2 (SEQ ID NO:24)- AISGSGGSTYYADSVKG CDR3 not by Kabat (SEQ ID NO:25) AKDGGYYDSGAGDY or CDR3 (SEQ ID NO:70) - DGGYYDSGAGDY CDR2 (SEQ ID NO:27) AASSLQS CDR3 (SEQ ID NO:28) QQGVSYPRT ADI-27749 (A49) EVQLVESGGGLVKPGGSLRLSCAASGF TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPMGAAA GWFDPWGQGTLVTVSS (SEQ ID NO:7) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) - SYSMN CDR2 (SEQ ID NO :30)SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO:31) ARGAPMGAAAGWFDP or CDR3 (SEQ ID NO:71) - GAPMGAAAGWFDP DIQMTQ SP S S VS AS VGDRVTI TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO:34) QQGVSFPRT ADI-29378 (E78) QVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYMHWVRQAPGQGLEWMGII NPSGGSTSYAQKFQGRVTMTRDTSTSTST VYMELSSLRSEDTAVYYCAREGAGFA YGMD YYYMD VWGKGTTVT VS S (SEQ ID NO:9) CDR1 not according to Kabat (SEQ ID NO :35) - YTFTSYYMH or CDR1 (SEQ ID NO:45) - SYYMH CDR2 (SEQ ID NO:36)- IINPSGGSTSYAQKFQG CDR3 not according to Kabat (SEQ ID NO :37) - AREGAGFAYGMDYYYMDV or CDR3 (SEQ ID NO :72)- EGAGFAYGMDYYYMDV EIVLTQSPATLSLSPGERATLS CRASQSVSSYLAWYQQKPGQ A PRLLI YD A S NR ATGIP A RF S GSGSGTDFTLTISSLEPEDFAV YYCQQ SDNWPF TFGGGTK VE IK (SEQ ID NO: 10) CDR1 (SEQ ID NO:38) RASQSVSSYLA CDR2 (SEQ ID NO:39) DASNRAT CDR3 (SEQ ID NO:40) QQSDNWPFT A49MQ EVQLVESGGGLVKPGGSLRLSCAASGF DIQMTQ SP S S VS AS VGDRVTI

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TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPQGAAA GWFDPWGQGTL VTVSS (SEQ ID NO:83) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) - SYSMN CDR2 (SEQ ID NO :30)SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO:73) ARGAPQGAAAGWFDP or CDR3 (SEQ ID NO:74) - GAPQGAAAGWFDP TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO:34) QQGVSFPRT A49ML EVQLVESGGGLVKPGGSLRLSCAASGF TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPLGAAA GWFDPWGQGTL VTVSS (SEQ ID NO:84) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) - SYSMN CDR2 (SEQ ID NO :30) SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO :75) ARGAPLGAAAGWFDP or CDR3 (SEQ ID NO :76) - GAPLGAAAGWFDP DIQMTQ SP S S VS AS VGDRVTI TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO:34) QQGVSFPRT A49MI EVQLVESGGGLVKPGGSLRLSCAASGF TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPIGAAA GWFDPWGQGTL VTVSS (SEQ ID NO:85) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) DIQMTQ SP S S VS AS VGDRVTI TCRASQGISSWLAWYQQKPG KAPKLLIY AAS SLQ SGVP SRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) -

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- SYSMN CDR2 (SEQ ID NO:30)- SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO:77) ARGAPIGAAAGWFDP or CDR3 (SEQ ID NO:78) - GAPIGAAAGWFDP RASQGISSWLA CDR2 (SEQ ID NO:33) - AASSLQS CDR3 (SEQ ID NO:34) - QQGVSFPRT A49MF EVQLVESGGGLVKPGGSLRLSCAASGF TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPFGAAA GWFDPWGQGTLVTVSS (SEQ ID NO:86) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) - SYSMN CDR2 (SEQ ID NO :30)SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO :79) ARGAPFGAAAGWFDP or CDR3 (SEQ ID NO:80) - GAPFGAAAGWFDP DIQMTQ SP S S VS AS VGDRVTI TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO 34) QQGVSFPRT A49MV EVQLVESGGGLVKPGGSLRLSCAASGF TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPVGAAA GWFDPWGQGTLVTVSS (SEQIDNO:41) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) -SYSMN CDR2 (SEQ ID NO :30) SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO:81) ARGAPVGAAAGWFDP or CDR3 (SEQ ID NO:82) - GAPVGAAAGWFDP DIQMTQ SP S S VS AS VGDRVTI TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO:34) QQGVSFPRT A49- EVQLVESGGGLVKPGGSLRLSCAASGF DIQMTQ SP S S VS AS VGDRVTI consensuses and I TF S S YSMNWVRQ APGKGLEW VS SIS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPXGAAA GWFDPWGQGTLVTVSS where X is M, L, I, V, Q or F (SEQ ID NO:42) CDR1 not by Kabat (SEQ ID NO:29) FTFSSYSMN or CDR1 (SEQ ID NO:48) - SYSMN CDR2 (SEQ ID NO :30) SISSSSSYIYYADSVKG CDR3 not by Kabat (SEQ ID NO:43) ARGAPXGAAAGWFDP or CDR3 (SEQ ID NO:44) - GAPXGAAAGWFDP where X represents M, L, I, V, Q or F TCRASQGISSWLAWYQQKPG KAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFA TYYCQQGVSFPRTFGGGTKV EIK (SEQ ID NO:8) CDR1 (SEQ ID NO:32) RASQGISSWLA CDR2 (SEQ ID NO:33) AASSLQS CDR3 (SEQ ID NO:34) QQGVSFPRT

One advantage of one or more of the antibody heavy chain variable domain amino acid sequences described above is that they can bind to human and cynomolgus NKG2D to agonize the receptor and compete with natural ligands for receptor binding. Additional antigen binding sites that bind NKG2D and have one or more of these properties are also particularly useful and can be identified using competition binding assays known in the art. For example, additional antigen-binding fragments can be identified by competition with

ADI-29379, ADI-29463, ADI-27744, ADI-27749, or ADI-29378 to bind to both human NKG2D and optionally cynomolgus NKG2D.

Another advantage of NKG2D binding regions that contain the antibody heavy chain variable domains and light chain variable domain sequences described above is that they can bind NKG2D with high affinity. In some embodiments, the NKG2D binding sites bind to NKG2D with a KD of 0.1 to 1000 nM. In some embodiments, the NKG2D binding sites bind to NKG2D with a KD of 1 to 500 nM. In some embodiments, the NKG2D binding sites bind to NKG2D with a K _D of 5 to 100 nM. In some embodiments, the NKG2D binding sites bind to NKG2D with a KD of 10 to 62 nM.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91%, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 2. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 1, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 11 or SEQ ID NO: 91, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 12, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 13 or 68. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 2, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 14, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 15, and the CDR3 sequence represented by amino acid sequence SEQ ID NO: 16.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 4. In some embodiments, the antigen binding region comprises an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 3, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 17 or SEQ ID NO: 92, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 18, and the CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 69. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 4, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 20, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 21, and the CDR3 sequence , represented by the amino acid sequence SEQ ID NO: 22.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 6. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 5, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 23 or SEQ ID NO: 93, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 24, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 70. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92 , 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 6, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 26, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 27, and the CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 7, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 71. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94 , 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 33, and the sequence CDR3 represented by the amino acid sequence SEQ ID NO: 34.

The amino acid residue M at position 102 of SEQ ID NO: 7, which is located in CDR3 of the heavy chain variable domain, may be mutated. In some embodiments, M102 is replaced with an uncharged moiety. In some embodiments, M102 is replaced with a hydrophobic moiety (Gly, Ala, Val, Leu, Ile, Pro, Phe, or Tip). In some embodiments, M102 is replaced with a polar residue (Ser, Thr, Cys, Asn, Gln, or Tyr). In some embodiments, M102 is substituted with Leu, Ile, Val, Gln, or Phe.

Accordingly, in certain embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 83, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96 , 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 83, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93 , 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 84, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, the antigen binding region comprises an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 84, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and the CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 75 or

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SEQ ID NO: 76. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains a CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, a CDR2 sequence represented by the amino acid sequence SEQ ID NO: 33, and a CDR3 sequence represented by the amino acid sequence SEQ ID NO: 34.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 85, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 85, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94 , 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 33, and the sequence CDR3 represented by the amino acid sequence SEQ ID NO: 34.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 86, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 86, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 79 or 80. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96 , 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 33, and the CDR3 sequence represented by the amino acid sequence sequence SEQ ID NO: 34.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 41, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 8. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 41, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 29 or SEQ ID NO: 94, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 30, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94 , 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 8, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 32, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 14 043960 33, and the CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 10. In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 9, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 35 or SEQ ID NO: 91, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 36, and the sequence CDR3 represented by the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 72. In some embodiments, an antigen binding region comprising an antibody light chain variable domain of at least 90% amino acid sequence (e.g., 91, 92, 93, 94 , 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 10, contains the CDR1 sequence represented by the amino acid sequence SEQ ID NO: 38, the CDR2 sequence represented by the amino acid sequence SEQ ID NO: 39, and the sequence CDR3 represented by the amino acid sequence SEQ ID NO: 40.

In some embodiments, the present invention provides an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 6, which does not block the binding of antibodies to NKG2D MS, 1D11 and MAB139 to NKG2D.

In embodiments, an antigen binding region comprising an antibody heavy chain variable domain that contains an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to that of sequences of SEQ ID NO: 7, 83, 84, 85, 86, or 87, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to SEQ ID NO: 8, does not block binding of an antigen binding region comprising an antibody heavy chain variable domain that contains at least 90% of the amino acid sequence (e.g., 91, 92, 93, 94 , 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and an antibody light chain variable domain that contains at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to SEQ ID NO: 6, with NKG2D.

In some embodiments, an antigen binding region comprising an antibody heavy chain variable domain that contains an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical amino acid sequence of SEQ ID NO: 5, and an antibody light chain variable domain that contains at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) of the amino acid sequence identical to SEQ ID NO: 6, bind to a unique epitope on NKG2D, unlike MS, 1D11, MAB139, ADI-27749 and F47-binding epitope(s).

Antibodies and multispecific binding proteins.

In some embodiments of the present invention, NKG2D antigen binding regions formed by pairing of an antibody heavy chain variable domain with a light chain variable domain described herein may be included in larger proteins, such as intact antibodies, multispecific binding proteins, or multispecific binding proteins. antibodies. For example, the NKG2D binding region may be combined with a second component, such as a second antigen binding region. In some embodiments, the second antigen-binding region binds to one or more tumor-associated antigens, such as CD33, HER2, EpCAM, CD2, CD3, CD8, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33 , CD37, CD38, CD40, CD45RO, CD48, CD52, CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CSAp, CA-125, TAG-72, EFGR/ERBB1, IGF1R, HER3 , HER4, IGF-1R, c-Met, PDGFR, MUC1, MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, TRAILR1, TRAILR2, Fas (CD95), DR3, DR4, DR5, DR6, VEGF, PIGF, tenascin, ED-B fibronectin, PSA and IL-6, MAGE-A3, B7.1, B7.2, CTLA4 or PD1. The binding of the multispecific binding protein to NKG2D and to the tumor-associated antigen on the cancer cell brings the cancer cell closer to the natural killer cell, which facilitates direct and indirect destruction of the cancer cell by the natural killer cell.

In some embodiments, in addition to the NKG2D binding region and the tumor-associated antigen binding region, the multispecific binding protein may further include a domain that binds to CD16, an Fc receptor on the surface of leukocytes, including natural killer cells macrophages, neutrophils, eosinophils, mast cells and follicular dendritic cells. In some embodiments, the CD16 binding domain may include the Fc region of an antibody or a portion thereof. In some embodiments, the domain that binds CD16 comprises the hinge domains, CH2 and CH3 domains of the antibody Fc region with or without a CH1 domain. In some embodiments, the Fc region of the antibody is derived from the Fc regions of human and/or other mammalian immunoglobulins. In the Fc region, CD16 binding is known to be mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mediated through the amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - Ile 332, Leu 234 - Ser 239 and the carbohydrate residue K-acetylN-glucosamine in the CH2 domain (see. , Sondermann et al., Nature, 406(6793):267-273). Based on known domains and amino acid residues, in some embodiments, mutations may be selected within the CD16 binding domain to increase or decrease its binding affinity to CD16. Selection methods are well known in the art, such as phage display libraries or yeast surface display cDNA libraries. Suitable selection methods can also be designed by one skilled in the art based on the known three-dimensional interaction structure.

The multispecific binding proteins described herein can take various formats. For example, one format is a heterodimeric multispecific antibody that includes a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second heavy chain domain CH1. The second immunoglobulin light chain includes a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen-binding site that binds tumor antigen. The first Fc domain and the second Fc domain together can bind CD16 (Fig. 1).

Another exemplary format includes a heterodimeric multispecific antibody that includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domain fused by either a linker or the hinge region of the antibody to a single chain variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain that pairs and binds with NKG2D. The second immunoglobulin heavy chain includes an Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The light chain of an immunoglobulin includes a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain combines with the immunoglobulin light chain and binds to the tumor-associated antigen. The first Fc domain and the second Fc domain together can bind CD16 (Fig. 2). Additional formats of multispecific binding proteins can be developed by combining the various formats of NKG2D binding fragments described herein.

One or more additional binding motifs may be fused to the C-terminus of the CH3 constant region domain, optionally via a linker sequence. In some embodiments, the antigen binding region 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 triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half-antibodies, each with one light and one heavy chain, which are derived from two parent antibodies. The triomab form is a heterodimeric construct containing ½ rat antibody and ½ mouse antibody.

In some embodiments, the multispecific binding protein is a common light chain (LC) form that uses knob-in-knot (KIH) technology. KIH involves the design of CH3 domains to create a knob or trench in each heavy chain to promote heterodimerization. The concept of Fc knobs-in-hollows (KIH) technology was to introduce a knob in one CH3 domain (CH3A) by replacing a small residue with

- 16 043960 volumetric (i.e. T366W _CH3A in EU numbering). To accommodate the overhang, an additional surface overhang was created on another CH3 domain (CH3B) by replacing the nearest adjacent residues to the overhang with smaller ones (i.e., T366S/L368A/Y407V _CH3B ). The knob mutation was optimized by screening a structure-targeted phage library (Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. ( 1997), 270(1):26-35). X-ray crystal structures of KIH Fc variants (Elliott JM, Ultsch M., Lee J., Tong R., Takeda K., Spiess C., et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2 -CH3 hydrophobic interaction. J. Mol. Biol. (2014), 426(9):1947-57; Mimoto F., Kadono S., Katada H., Igawa T., Kamikawa T., Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol. Immunol. (2014), 58(1):132-8) demonstrated that heterodimerization is thermodynamically supported by hydrophobic interactions caused by steric complementarity at the interface between the core of the CH3 domain and the internal surface, whereas the valley-valve and projection-ridge surfaces are not favorable for homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

In some embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™), which combines the target binding domains of two monoclonal antibodies through flexible natural linkers to produce a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which the variable domain-targeting antigen 2 is fused to the N-terminus of the Fab-targeting antigen 1 variable domain. The design contains normal Fc.

In some embodiments, the multispecific binding protein is in the form of an orthogonal surface Fab (Ortho-Fab), which is a heterodimeric construct containing two Fabs binding to target 1 and target 2 fused to an Fc. LCHC pairing is ensured by an orthogonal surface. Heterodimerization is mediated by mutations in Fc. In an approach to ortho-Fab IgG (Lewis SM, Wu X., Pustilnik A., Sereno A., Huang F., Rick HL, et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat Biotechnol (2014), 32(2):191-8), structure-based regional design introduces complementary mutations into the LC and HC _vh surface. _ch1 in only one Fab, without any changes in the other Fab.

In some embodiments, the multispecific binding protein is in a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in the form of an ES, which is a heterodimeric construct containing two different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is mediated by electrostatic mutation in Fc.

In some embodiments, the multispecific binding protein is in the form of a κλ body, which are heterodimeric constructs with two different Fabs fused to an Fc, stabilized by heterodimerization mutations: Fab-targeting antigen 1 contains kappa-LC, while the second Fab-targeting antigen 2 contains lambda-LC.

In some embodiments, the multispecific binding protein is in the form of a Fab exchange (antibodies that exchange Fab fragments by exchanging the heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies). The Fab fragment exchange form (cFae) is a heterodimer containing two target-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations.

In some embodiments, the multispecific binding protein is in the form of a SEED body that is a heterodimer containing two target-binding Fabs 1 and 2 and Fcs stabilized by heterodimerization mutations. The chain-exchange engineered domain (SEED) has been developed to generate asymmetric and bispecific antibody-like molecules, allowing for expanded therapeutic applications of natural antibodies. This protein engineering platform is based on the exchange of structurally related immunoglobulin sequences in conserved CH3 domains. The SEED design allows efficient generation of AG/GA heterodimers while preventing homodimerization of the AG and GA SEED CH3 domains. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

In some embodiments, the multispecific binding protein is in the form of LuZ-Y, in which a leucine zipper is used to induce heterodimerization of two different HCs. (Wranik, B.J. et al., J. Biol. Chem. (2012), 287:43331-9). The LuZ-Y form is a heterodimer containing two different target-binding scFabs 1 and 2 fused to an Fc. Heterodimerization is mediated by leucine zipper motifs fused to the C terminus of the Fc.

In some embodiments, the multispecific binding protein is in the form of a Cov-X-Body (in bispecific CovX antibodies, two different peptides are linked together using a branched azetidinone linker and fused to the framework antibody under mild conditions in a site-specific manner). While pharmacophores are responsible for functional activity, the antibody scaffold provides a long half-life and Ig-like distribution. Pharmacophores can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies. (Doppalapudi V.R. et al., PNAS (2010),

107(52); 22611-22616).

In some embodiments, the multispecific binding protein is in a heterodimeric Oasc-Fab format that includes Fab binding to target 1 and scFab binding to target 2 fused to an Fc. Heterodimerization is mediated by mutations in Fc.

In some embodiments, the multispecific binding protein is in a DuetMab format containing two different Antigen 1 and 2 binding Fabs and Fcs stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure proper pairing of LC and NS.

In some embodiments, the multispecific binding protein is in the CrossmAb format, which is a heterodimeric construct with two different target binding Fabs 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are swapped, eg CH1 is connected to VL while CL is connected to VH.

In some embodiments, the multispecific binding protein is in the CrossmAb format, which are homodimeric constructs where a Fab binding to antigen 2 is fused to the N-terminus of an HC Fab that binds to antigen 1. The construct contains a wild-type Fc.

Heavy chains of heterodimeric antibodies.

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 the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. Preferential assembly of heterodimeric heavy chains in the multispecific binding proteins described herein can be achieved by including different pairs of amino acid substitutions in the first CH3 domain in the first heavy chain polypeptide and in the second CH3 domain in the second heavy chain polypeptide, allowing the two chains to selectively heterodimerize with each other, as shown in US 13/494870, US 16/028850, US 11/533709, US 12/875015, US 13/289934, US 14/773418, US 12/811207, US 13/866756, US 14 /647480, US 14/830336. In some embodiments, the multispecific binding proteins comprise a human IgG1 Fc domain. Below are various examples of amino acid substitutions in a pair of human IgG1 Fc domains to facilitate heterodimerization of the two heavy chains. Each amino acid substitution position is numbered according to the EU index, as in Kabat.

In one scenario, the amino acid substitution in the first polypeptide replaces the parent 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 parent amino acid (s) smaller amino acid(s) selected from alanine (A), serine (S), threonine (T) or valine (V), such that the larger amino acid substitution (protrusion) fits into the surface of the smaller amino acid substitutions (cavity) . For example, one polypeptide may include the T366W substitution, and another may include three substitutions, including T366S, L368A, and Y407V.

Alternatively, amino acid substitutions can be selected from the following sets of substitutions shown in table. 2.

- 18 043960

table 2

First polypeptide Second polypeptide Set 1 S364E/F405A Y349K/T394F Set 2 S364H/D401K Y349T/T411E Set 3 S364H/T394F Y349T/F405A Set 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6 S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357Q L368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11 K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370S S364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

Alternatively, amino acid substitutions can be selected from the following sets of substitutions shown in table. 3.

Table 3

First polypeptide Second polypeptide Set 1 K409W D399V/F405T Set 2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set 5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409W E357W/D399V/F405T

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

Table 4

First polypeptide Second polypeptide Set 1 T366K/L351K L351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349D Set 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368E Set 6 E356K/D399K K392D/K409D

Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from table. 5.

Table 5

First polypeptide Second polypeptide L351Y, D399R, D399K, S400K, S400R, Y407A, Y407I, Y407V T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

Alternatively, the at least one amino acid substitution may be selected from the following set of substitutions in table. 6, wherein the amino acid at the position(s) indicated in the First Polypeptide column is replaced by any known negatively charged amino acid, and the amino acid at the position(s) indicated in the Second Polypeptide column is replaced by any known positively charged amino acid.

Table 6

First polypeptide Second polypeptide KZ 92, KZ 70, K409 or K439 D399, E356 or E357

- 19043960

Alternatively, the at least one amino acid substitution may be selected from the following set in table. 7, wherein the amino acid at the position(s) indicated in the First Polypeptide column is replaced by any known positively charged amino acid, and the amino acid at the indicated position(s) in the Second Polypeptide column is replaced by any known negatively charged amino acid.

Table 7

First polypeptide Second polypeptide D399, E356 or E357 K409, K439, KZ 70 or KZ 92

Alternatively, amino acid substitutions can be selected from the following set of substitutions shown in table. 8.

Table 8

First polypeptide Second polypeptide T350V, L351Y, F405A and Y407V T35OV, T366L, K392L and T394W

Alternatively or in addition, the structural stability of heterodimeric heavy chains in multispecific binding proteins can be increased by introducing S354C on 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 binding proteins described above can be produced using recombinant DNA technology well known to one 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 the first immunoglobulin light chain may be cloned into a third expression vector; a fourth nucleic acid sequence encoding a second immunoglobulin light chain may be cloned into a fourth expression vector; wherein the first, second, third and fourth expression vectors can be stably transfected together into host cells to produce multimeric proteins.

To achieve the highest yield of multispecific binding proteins, different ratios of the first, second, third and fourth expression vectors can be examined to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated to generate a cell bank using methods known in the art, such as limited dilution, ELISA, FACS, microscopy or Clonepix.

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

The protein contains an antigen-binding site that competes with the NKG2D-binding sites described herein.

In certain embodiments, the present invention provides a protein comprising an antigen binding site that competes with the NKG2D binding sites described herein for binding to NKG2D. The NKG2D binding regions described herein contain the amino acid sequences of SEQ ID NO: 1 and 2; amino acid sequences SEQ ID NO: 3 and 4; amino acid sequences SEQ ID NO: 5 and 6; amino acid sequences SEQ ID NO: 7 and 8; amino acid sequences SEQ ID NO: 9 and 10; amino acid sequences SEQ ID NO: 83 and 8; amino acid sequences SEQ ID NO: 84 and 8; amino acid sequences SEQ ID NO: 85 and 8; amino acid sequences SEQ ID NOs: 86 and 8 or amino acid sequences SEQ ID NOs: 87 and 8. These NKG2D-binding regions can bind to various epitopes on NKG2D mapped by surface plasmon resonance. For example, unlike ADI-27749 and other existing anti-NKG2D antibodies, ADI-27744 binds to a different epitope on NKG2D, as demonstrated in Example 2.

In some embodiments, the antigen binding region of a protein that competes with NKG2D binding regions includes a heavy chain variable domain having at least 50% amino acid sequence (e.g., 50, 60, 70, 80, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence that is at least 50% identical (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, an antigen binding region of a protein that competes with NKG2D -binding regions, includes a heavy chain variable domain having at least 50% amino acid sequence (for example, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain having an amino acid sequence that is at least 50% identical (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an antigen binding region of a protein that competes with NKG2D binding regions includes a heavy chain variable domain having an amino acid sequence of at least at least 50% (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and variable domain light chain having an amino acid sequence at least 50% (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence SEQ ID NO: 6. In some embodiments, the antigen binding region of the protein that competes with NKG2D binding regions includes a heavy chain variable domain having an amino acid sequence of at least 50% (e.g., 50, 60, 70, 80, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical in amino acid sequence to SEQ ID NO: 7, and a light chain variable domain having an amino acid sequence that is at least 50% identical (e.g. 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antigen binding region of the protein that competes with NKG2D-binding regions, includes a heavy chain variable domain having at least 50% amino acid sequence (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain having an amino acid sequence of at least 50% (e.g., 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the protein comprising an antigen binding site that competes with the NKG2D binding sites described herein further includes a second antigen binding site that binds a tumor-associated antigen and/or a CD16 binding site. In some embodiments, the CD16 binding site is an antibody constant region or portion thereof capable of binding CD16. In some embodiments, the CD16 binding site comprises a human IgG1 Fc domain.

Protein expression cell.

In one aspect, the present invention provides a cell containing one or more nucleic acids encoding a protein that contains: an NKG2D binding region with a heavy chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93 , 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence that is at least 90% identical (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 2; An NKG2D binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO : 3, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 4; An NKG2D binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO : 5, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 6; An NKG2D binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO : 7, 83, 84, 85, 86 or 87, and a light chain variable domain having at least 90% amino acid sequence (for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence SEQ ID NO: 8; or an NKG2D binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO : 10.

Therapeutic applications.

The invention relates to methods for enhancing tumor cell death and/or treating cancer using a multispecific binding protein described in the present invention and/or

- 21 043960 pharmaceutical composition described in the invention. The methods can be used to treat various types of cancer. The type of cancer to be treated desirably matches the type of cancer cell to which the protein binds. Additional aspects and embodiments of the therapeutic methods are described below.

Pharmaceutical compositions.

In one aspect, the present disclosure also provides pharmaceutical compositions that contain an effective amount of a protein that contains an NKG2D binding site described herein, or an NKG2D binding site that competes with NKG2D binding sites described herein, and pharmaceutically acceptable medium.

In some embodiments, the composition comprises a protein that contains an antigen binding region with a heavy chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 %) identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% ) identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the composition contains a protein that includes an antigen binding region with a heavy chain variable domain having at least 90% amino acid sequence (e.g., 91, 92, 93, 94, 95 , 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain having an amino acid sequence that is at least 90% identical (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the composition contains a protein that includes an antigen binding region with a heavy chain variable domain having at least 90% amino acid sequence (e.g. , 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical in amino acid sequence to SEQ ID NO: 5, and a light chain variable domain having an amino acid sequence that is at least 90% identical (e.g. 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the composition contains a protein that includes an antigen binding region with a heavy chain variable domain having an amino acid a sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, 83, 84, 85, 86, or 87 , and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the composition contains a protein that includes an antigen binding region with a heavy chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 %) identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% ) identical to the amino acid sequence of SEQ ID NO: 10.

The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., ^17th ed., 1985. For a brief review of drug delivery methods, see, for example, Langer (Science, 249:1527-1533 , 1990).

For example, the present disclosure may exist in the form of an aqueous pharmaceutical composition comprising a therapeutically effective amount of protein in a buffered solution forming the composition. Aqueous vehicles may include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In certain embodiments, an aqueous formulation comprising a protein disclosed herein is prepared in a pH buffered solution. The pH of the preparations is usually from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 7 to 8, for example from 7 to 7.5. Ranges intermediate to the above pH values should also be part of this disclosure. For example, value ranges that use a combination of any of the above values as an upper and/or lower limit are intended to be included. Examples of buffers that will adjust the pH in this range include acetate (such as sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), about 1.5 mg/mL disodium phosphate

- 22 043960 dihydrate (for example, 1.53 mg/ml), about 0.9 mg/ml sodium dihydrogen phosphate dihydrate (for example, 0.86 mg/ml) and about 6.2 mg/ml sodium chloride (for example, 6.165 mg /ml). In some embodiments, the buffer system includes 1 to 1.5 mg/mL citric acid, 0.25 to 0.5 mg/mL sodium citrate, 1.25 to 1.75 mg/mL disodium phosphate dihydrate, 0. 7 to 1.1 mg/ml sodium dihydrogen phosphate dihydrate and 6.0 to 6.4 mg/ml sodium chloride. The pH of the liquid formulation can be adjusted by adding 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 some embodiments, the formulation includes an aqueous carrier that is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing a liquid formulation. Exemplary vehicles include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

A polyol that acts as a tonic and can stabilize the antibody may also be included. The polyol is added to the formulation in an amount that may vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous composition may be isotonic. The amount of polyol added can also be varied relative to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (such as mannitol) may be added compared to a disaccharide (such as trehalose). In some embodiments, the polyol that can be used in the composition as a tonicity control agent is mannitol. In certain embodiments, the concentration of mannitol may be from about 5 to about 20 mg/ml. In certain embodiments, the concentration of mannitol may be about 7.5-15 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, a sorbitol polyol may be included in the composition.

A detergent or surfactant may also be added to the formulation. Typical detergents include nonionic detergents such as polysorbates (eg, polysorbates 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of particles in the composition and/or reduces adsorption. In certain embodiments, the composition may include a surfactant that is a polysorbate. In some embodiments, the composition may contain the detergent polysorbate 80 or Tween 80. Tween 80 is the term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4 ^th edi., 1996). In some embodiments, the formulation may contain from about 0.1 to about 10 mg/ml or from about 0.5 to about 5 mg/ml polysorbate 80. In some embodiments, about 0.1% polysorbate 80 may be added to the composition.

In certain embodiments, the liquid composition of the present invention can be formulated as a solution at a concentration of 10 mg/ml in combination with sugar at stabilizing levels. In certain embodiments, the liquid composition may be formulated in an aqueous carrier. In some embodiments, the stabilizer may be added in an amount no greater than that which would result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative, which is added to the compositions of the present invention to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a reusable (reusable) formulation.

In some embodiments, the present disclosure relates to an extended shelf life formulation comprising a protein of the present invention in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water and sodium hydroxide .

Deamidation is a common variant of peptides and proteins that can occur during fermentation, cell collection/clarification, purification, drug/drug storage, and during sample analysis. Deamidation is the loss of NH3 from a protein, forming a succinimide intermediate that can undergo hydrolysis. An intermediate content of succinimide leads to a decrease in the mass of the initial peptide by 17 Da. Subsequent hydrolysis leads to an increase in mass by 18 units. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. Thus, deamidation is usually defined as an increase in mass of 1 unit. Deamidation of asparagine results in aspartic or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues located

- 23 043960 close to Asn in the peptide chain, affect the rate of deamidation. Gly and Ser following Asn in protein sequences result in higher susceptibility to deamidation. In certain embodiments, the liquid composition of the present invention may be maintained under pH and humidity conditions to prevent deamination of the protein product.

In some embodiments, the composition is a lyophilized composition. In certain embodiments, the formulation is freeze-dried (lyophilized) and is contained in about 12-60 vials. In some embodiments, the formulation is lyophilized, and 45 mg of the lyophilized formulation may be contained in one vial. In certain embodiments, about 40-100 mg of the lyophilized formulation is contained in one vial. In some embodiments, a lyophilized formulation of 12, 27, or 45 vials is combined to produce a therapeutic dose of protein in an intravenous dosage form. The composition may be a liquid composition. In some embodiments, the liquid formulation is stored in an amount of from about 250 to about 1000 mg/vial. In certain embodiments, the liquid formulation is stored in an amount of about 600 mg/vial. In certain embodiments, the liquid formulation is stored in an amount of about 250 mg/vial.

In some embodiments, the lyophilized composition includes proteins described in the present invention 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 also include one or more of a buffering agent, a surfactant, an excipient and/or a preservative. The amount of sucrose or maltose suitable for stabilizing the lyophilized drug may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In some embodiments, the protein/sucrose or maltose weight ratio may be from 1:2 to 1:5.

In some embodiments, the pH of the formulation may be adjusted prior to lyophilization by adding a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide. Before lyophilization, the pH of the solution containing the protein of the present invention may be adjusted to a range of 6 to 8. In some embodiments, the pH range for the lyophilized drug may be 7 to 8.

In certain embodiments, filler may be added. An excipient is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized mass (eg, facilitates the production of a substantially uniform lyophilized mass that maintains an open-cell structure). Exemplary excipients include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized compositions of the present invention may contain such excipients.

In some embodiments, the lyophilized protein product is formulated in an aqueous vehicle. The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffer solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In certain embodiments, the lyophilized drug of the present invention is reconstituted with either Sterile Water for Injection, USP or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder is dissolved in the solution. In some embodiments, the lyophilized protein product of the present invention consists of about 4.5 ml of water for injection and is diluted with 0.9% saline (sodium chloride solution).

The protein compositions can be sterilized by conventional sterilization methods or can be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The resulting compositions in solid form can be packaged in multiple single-dose containers, each containing a fixed amount of the above agent or agents. The composition in solid form may also be packaged in a variable quantity container.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be adjusted to provide an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without being toxic to the patient.

The specific dose may be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, the patient's dose may be tailored to the patient's approximate body weight or surface area. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Those skilled in the art will routinely further refine the calculations necessary to determine the appropriate dosage for treatment, especially in light of the dosage information and tests disclosed herein. Dosage can also be determined by using known dose determination assays used in conjunction with appropriate dose-response data. An individual patient's dosage may be adjusted as disease progression is monitored. Blood levels of the target construct or complex in a patient can be measured to see if dosage adjustments need to be made to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which target constructs and/or complexes and their dosages are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta, 308:43-53, 2001; Steimer et al., Clinica Chimica Acta, 308:33-41, 2001).

In general, dosages based on body weight range from about 0.01 μg to about 100 mg/kg body weight, for example, from about 0.01 μg to about 100 mg/kg body weight, from about 0.01 µg to about 50 mg/kg body weight, from about 0.01 µg to about 10 mg/kg body weight, from about 0.01 µg to about 1 mg/kg body weight, from about 0.01 to about 100 µg/kg body weight, from about 0.01 to about 50 μg/kg body weight, from about 0.01 to about 10 μg/kg body weight, from about 0.01 to about 1 μg/kg body weight, from about 0.01 to about 0.1 μg/kg body weight, from about 0.1 μg to about 100 mg/kg body weight, from about 0.1 μg to about 50 mg/kg body weight, from about

0.1 mcg to about 10 mg/kg body weight, from about 0.1 mcg to about 1 mg/kg body weight, from about

0.1 to about 100 μg/kg body weight, from about 0.1 to about 10 μg/kg body weight, from about

0.1 to about 1 μg/kg body weight, from about 1 μg to about 100 mg/kg body weight, from about μg to about 50 mg/kg body weight, from about 1 μg to about 10 mg/kg body weight, from about 1 μg to about 1 mg/kg body weight, from about 1 to about 100 μg/kg body weight, from about 1 to about 50 μg/kg body weight, from about 1 to about 10 μg/kg body weight, from about 10 μg to about 100 mg/kg body weight, from about 10 μg to about 50 mg/kg body weight, from about μg to about 10 mg/kg body weight, from about 10 μg to about 1 mg/kg body weight, from about to about 100 µg/kg body weight, from about 10 to about 50 µg/kg body weight, from about 50 µg to about 100 mg/kg body weight, from about 50 µg to about 50 mg/kg body weight, from about µg to about 10 mg/kg body weight, from about 50 µg to about 1 mg/kg body weight, from about to about 100 µg/kg body weight, from about 100 µg to about 100 mg/kg body weight, from about 100 mcg to about 50 mg/kg body weight, from about 100 mcg to about 10 mg/kg body weight, from about 100 mcg to about 1 mg/kg body weight, from about 1 to about 100 mg/kg body weight, from about 1 to about 50 mg/kg body weight, from about 1 to about 10 mg/kg body weight, from about 10 to about 100 mg/kg body weight, from about 10 to about 50 mg/kg body weight, from about 50 up to about 100 mg/kg body weight.

Doses may be administered one or more times daily, weekly, monthly or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the repetition frequency for dosing based on the measured residence times and concentrations of the target construct or complex in body fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter, or by direct injection into the lesion. They 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.

Enhancing tumor cell death and cancer treatment.

The invention relates to methods for enhancing tumor cell death and/or treating cancer in a patient. In some embodiments, the method includes targeting tumor cells and natural killer cells with a multispecific binding protein disclosed herein. In some embodiments, the method includes administering to a patient in need thereof a therapeutically effective amount of a protein and/or a desired composition thereof as described herein. In such embodiments, the multispecific binding protein may comprise an antigen binding region with a heavy chain variable domain having an amino acid sequence of at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% ) identical to the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain having an amino acid sequence of at least 90% (for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 2; an antigen binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 , and a light chain variable domain having an amino acid sequence at least 90% (eg, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 4; antigen binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to that of

- 25 043960 of the sequence SEQ ID NO: 5, and a light chain variable domain having an amino acid sequence of at least 90% (for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) identical to the amino acid sequence of SEQ ID NO: 6; an antigen binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 , 83, 84, 85, 86 or 87, and a light chain variable domain having an amino acid sequence of at least 90% (for example, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% ) identical to the amino acid sequence of SEQ ID NO: 8; or an antigen binding region with a heavy chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain having an amino acid sequence at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identical to the amino acid sequence of SEQ ID NO: 10 .

The type of cancer to be treated desirably matches the type of cancer cell to which the multispecific binding protein described herein binds. For example, treatment of epithelial cell adhesion molecule (EpCAM)-expressing cancer, such as colon cancer expressing EpCAM, is desirably treated using a multispecific binding protein described herein that binds EpCAM and NKG2D.

In some embodiments, the patients to be treated contain cancer cells that express one or more of the following: CD33, HER2, CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3 , HER4/ERBB4, MUC1, CEA, sMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-AZ, B7.1, B7.2, CTLA4 and PD1. In some embodiments, the patients to be treated have a solid cancer, such as brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colon cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, colorectal cancer, kidney cancer, stomach cancer, testicular cancer or uterine cancer. In other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal rectal cancer, anal cancer, rectal cancer, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, cancer bile duct, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, neuroendocrine tumors, cholangiocarcinoma, chondrosarcoma, choroid papilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, digestive system cancer, duodenal cancer, endocrine system cancer, endodermal tumor, endometrial hyperplasia, uterine stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbital cancer, cancer of the female genital organs, focal nodular hyperplasia, gall cancer bladder, gastric antrum cancer, gastric vault cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, liver adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, small bowel cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell cancer, colon cancer, leiomyosarcoma, lentigo melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, small cell cancer, oligodendroglial cancer, oral cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumors, plasmacytoma, pseudosarcoma, lung 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, spine cancer, squamous cell cancer, striated muscle cancer, submesothelial cancer, superficial spreading melanoma, T -cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary system cancer, cervical cancer, uterine body cancer, uveal melanoma, vaginal cancer, verrucous carcinoma VIPoma, vulvar cancer, well-differentiated carcinoma or tumor Wilms.

In some embodiments, the patients being treated have non-Hodgkin's disease

- 26 043960 lymphoma, such as B-cell lymphoma or T-cell lymphoma. In some 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 cell lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal B-cell lymphoma marginal zone cell lymphoma, nodal marginal zone cell 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 some other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, T - enteropathic cell lymphoma, subcutaneous panniculitis type T-cell lymphoma, anaplastic large cell lymphoma or peripheral T-cell lymphoma.

In some embodiments, the proteins described herein are used in combination with additional therapeutic agents to treat patients with cancer. Typical therapeutic agents that may be used as part of combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronit, methotrexate, doxorubicin, carbocon, pentostatin, nitractin, zinostatin , cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxan, nedaplatins, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxyfluridine, etretinate, isotretino in, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sisofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocytabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostat, epithiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diphytox, interleukin-2, luteinizing hormone releasing factor and variations of the above agents that may exhibit differential binding to the corresponding cognate receptor, and also increase or decrease serum half-life.

An additional class of agents that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Typical immune checkpoint inhibitors include agents that inhibit one or more of (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) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for the treatment of melanoma.

However, other agents that may be used as part of combination therapy in the treatment of cancer are monoclonal antibodies that target non-checkpoint targets (eg, Herceptin) and non-cytotoxic agents (eg, tyrosine kinase inhibitors).

Still other categories of anticancer agents include, for example, (i) an inhibitor selected from an ALK inhibitor, an ATR inhibitor, an A2A antagonist, a DNA base excision repair inhibitor, a Bcr-Abl tyrosine kinase inhibitor, a Bruton's tyrosine kinase inhibitor, a CDC7 inhibitor, a CHK1 inhibitor, a cyclin-dependent kinase inhibitor. , DNA-PK inhibitor, DNA-PK mTOR inhibitor, DNMT1 inhibitor, DNMT1 inhibitor plus 2-chlorodeoxyadenosine, HDAC inhibitor, Hedgehog signaling pathway inhibitor, IDO inhibitor, JAK inhibitor, mTOR inhibitor, MEK inhibitor, MELK inhibitor, MTH1 inhibitor, 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 OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25 or ICOS agonist; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF and G-CSF.

The proteins of the present invention can also be used as an adjunct to surgical removal of the primary tumor site.

The amount of protein and additional therapeutic agent and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering combination therapy to a patient in need of such administration, the therapeutic agents in combination or the pharmaceutical composition or compositions containing the therapeutic agents may be administered in any order, such as, for example, sequentially, simultaneously, together, simultaneously, and the like. In addition, for example, a protein described herein can be administered during the time that the additional therapeutic agent(s) are exerting their prophylactic or therapeutic effects, or vice versa.

The above description describes many aspects and embodiments of the present invention. The patent application specifically addresses all combinations and variations of aspects and

- 27 043960 embodiments.

Examples

The present invention, as generally described above, will now be more readily understood with reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1: Binding affinity of different NKG2D binding domains.

The kinetics and affinities of various NKG2D binding domains were assessed by surface plasmon resonance using a Biacore 8K instrument (GE Healthcare). Anti-human Fc antibody was immobilized onto the CM5 chip using standard amine coupling chemistry. Human monoclonal antibodies containing various NKG2D-binding domains were captured on the anti-human Fc chip at a density of approximately 100 pfu. Solutions containing 0.411–100 nM soluble mouse NKG2D human Fc dimers were injected over captured anti-NKG2D antibodies and control surfaces at 30 μl/min at 37°C. Surfaces were regenerated between cycles with a rapid injection of 10 mM glycine, pH 1.8. To obtain kinetic rate constants, double-reference data were fitted to a 1:1 interaction model using Biacore 8K Evaluation software (GE Healthcare). The binding equilibrium constant K _D was determined as the ratio of the dissociation constant kd and the association constant k _a (k _d /k _a ). As shown in table. 9 and FIG. 3, the binding affinity of NKG2D binding domains to NKG2D is in the range of 10-62 nM.

Table 9

NKG2D-binding domain k _a (1/Mc) kd(1/c) K _D (nM) ADI-27744 (A44) 2.95E+05 2.99E-03 10.1 ADI-27749 (A49) 3.95E+05 4.89E-03 12.4 ADI-29378 (E78) 8.32E+05 4.87E-02 58.5 ADI-29379 (E79) 4.43E+05 2.25E-02 50.7 ADI-29463 (F63) l,64E+06 l,01E-01 61.8

Example 2: Epitope-specific binding sorting of clone ADI-27744.

Sorting of the NKG2D binding domain of ADI-27744 (A44) was performed against a series of antibodies and ULBP6 (a natural ligand of NKG2D) by surface plasmon resonance using a Biacore 8K instrument. Briefly, mouse Fc-human NKG2D was captured using anti-mouse Fc antibody immobilized on a CM5 chip at a density of approximately 100 FU. This was followed by sequential injections of antibodies including anti-NKG2D monoclonal antibody containing ADI-27744, ADI-27749, F47 (sequences listed below) or 1D11 (commercial anti-NKG2D monoclonal antibody), ULBP6 (sequence listed below), MS (anti-NKG2D antibody from Novo Nordisk, sequences listed below) and MAB139 (anti-NKG2D antibody from R&D system, clone 149810) at 30 μl/min at 25°C. Biacore 8K software was used to analyze all data.

Table 10

Heavy chain variable region Light chain variable region F47 QVQLQQWGAGLLKPSETLSLTCAVYG GSF SGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSL KL S S VT AADT A VYYC ARARGPW SFDP WGQGTLVTVSS (SEQIDNO:51) CDR1 (SEQ ID NO:52) - GSFSGYYWS CDR2 (SEQIDNO:53) EIDHSGSTNYNPSLKS CDR3 (SEQ ID NO 54) ARARGPWSFDP DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLL IYKASSLESGVPSRFSGSGSGTEFT LTISSLQPDDFATYYCQQYDTFITF GGGTKVEIK (SEQIDNO:55) CDR1 (SEQ ID NO:56) RASQSISSWLA CDR2 (SEQ ID NO:57) - KASSLES CDR3 (SEQ ID NO:58) - QQYDTFIT MS QVHLQESGPGLVKPSETLSLTCTVSDD EIVLTQSPGTLSLSPGERATLSCRA

- 28 043960

SISSYYWSWIRQPPGKGLEWIGHISYS GS ANYNPSLKSRVTIS VDT SKNQF SLK LS SVTAADTAVYYC ANWDDAFNIWG QGTMVTVSS (SEQIDNO:59) CDR1 (SEQ ID NO:60) - SYYWS CDR2 (SEQ ID NO:61) HISYSGSANYNPSLKS CDR3 (SEQ ID NO:62) - WDDAFNI SQSVSSSYLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFT LTISRLEPEDF AVYYCQQ YGS SPW TFGGGTKVEIK (SEQ ID NO:63) CDR1 (SEQ ID NO:64) - RASQSVSSSYLA CDR2 (SEQ ID NO:65) - GASSRAT CDR3 (SEQ ID NO:66) - QQYGSSPWT

Amino acid sequence of ULBP SEQ ID NO: 67:

RRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGK

KLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSG SWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWL EDFLMGMDSTLEPSAGAPLAMSSG

Fig. 4A shows the injection profile of an anti-NKG2D monoclonal antibody containing ADI-27744 over immobilized NKG2D followed by injection of ULBP6.

Fig. 4B shows the injection profile of ULBP6 over immobilized NKG2D followed by injection of an anti-NKG2D monoclonal antibody including ADI-27744. These results indicate that anti-NKG2D monoclonal antibodies, including the antigen binding site of ADI-27744, do not block ULBP6 binding to NKG2D, i.e. ADI-27744 binds to a different epitope on NKG2D relative to ULBP6.

Fig. 4C shows the injection profile of MS monoclonal antibody over NKG2D followed by injection of ULBP6. Monoclonal antibody MS blocks the binding of ULBP6 to NKG2D.

Fig. 4D-4F show the injection profile of MS, 1D11, or MAB139 over immobilized NKG2D followed by injection of an anti-NKG2D monoclonal antibody containing ADI-27744.

Fig. 4G-4H show the injection profile of an anti-NKG2D monoclonal antibody containing ADI-27744 over immobilized NKG2D followed by an injection of an anti-NKG2D monoclonal antibody containing ADI-27749 or F47. ADI-27744 does not block the binding of MS, 1D11 and MAB139 to NKG2D. ADI-27749 and F47 do not block the binding of ADI-27744 to NKG2D. These results indicate that ADI-27744 binds to a unique epitope on NKG2D, unlike MS, 1D11, MAB139, ADI-27749, and F47-binding epitope(s).

Example 3 Trispecific binding proteins bind to NKG2D.

Mouse lymphoma EL4 cell lines were engineered to express human NKG2D. Trispecific binding proteins (TriNKET), each of which contains an NKG2D binding domain, a tumor-associated antigen binding domain (such as a CD33 or HER2 binding domain), and an Fc domain that binds CD16, as shown in FIG. 1 were analyzed for their affinity for extracellular NKG2D expressed on EL4 cells. Binding of multispecific binding proteins to NKG2D was determined using fluorophore-conjugated secondary anti-human IgG antibodies. Cells were analyzed by flow cytometry and the ratio of signal over background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

TriNKETs analyzed include CD33-TriNKET-A44 (ADI-27744 and CD33 binding domain), CD33-TriNKET-A49 (ADI-27749 and CD33 binding domain), CD33-TriNKET-F63 (ADI-29463 and CD33 binding domain), HER2 -TriNKET-A44 (ADI-27744 and CD33-binding domain), HER2-TriNKET-A49 (ADI-27749 and HER2-binding domain), HER2-TriNKET-F63 (ADI-29463 and HER-binding domain), and HER2- TriNKET-E79 (ADI-29379 and HER2-binding domain). The HER2-binding domain consisted of a heavy chain variable domain and a light chain variable domain of trastuzumab. The CD33 binding domain consisted of a heavy chain variable domain and a light chain variable domain as indicated below.

- 29 043960

SEQIDNO:49

OVOLVOSGAEVKKPGASVKVSCKASGYTFTDYVVHWVRQAPGOGLEWMGYINPYND

CDR1

GTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYRYEVYGMDYWGQ

CDR2 CDR3

GTLVTVSS

SEQIDNO:50

DIVLTOSPASLAVSPGORATICTASSSVNYIHWYOQKPGQPPKLLIYDTSKVASGVPAR

CDR1 CDR1

FSGSGSGTDFTLTINPVEANDTANYYCQQWRSYPLTFGQGTKLEIK

CDR3

All TriNKETs bind NKG2D on the surface of EL4 cells, but with different affinities. CD33-TriNKET-A44 shows the same binding profile as HER2-TriNKET-A44, as does CD33-TriNKET-A49, as does HER2-TriNKET-A49, and CD33-TriNKET-F63 for HER2-TriNKET-F63. NKG2D binding affinity for each clone was similar between cells expressing human and mouse NKG2D (Figs. 5, 6).

Example 4 Trispecific binding proteins bind to human tumor antigens.

Trispecific binding proteins bind to CD33.

The human AML cell line MV4-11, expressing CD33, was used to analyze TriNKET binding to tumor-associated antigen. TriNKET and the parental anti-CD33 monoclonal antibody were incubated with cells and binding was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and the ratio of signal over background (FOB) was calculated using the mean fluorescence intensity (MFI) from TriNKET and the parental anti-CD33 monoclonal antibody normalized to control secondary antibodies.

CD33-TriNKET-A44, CD33-TriNKET-A49, and CD33-TriNKET-F63 showed comparable levels of binding to CD33 compared to the parental CD33 antibody (Fig. 7).

Trispecific binding proteins bind to HER2.

Human cancer cell lines expressing HER2 were used to analyze the binding of TriNKET to tumor-associated antigen. The 786-O kidney cancer cell line expresses low levels of HER2, and the NCI-H661 human lung cancer cell line expresses moderate levels of HER2. TriNKET and optionally the parental anti-HER2 monoclonal antibody (trastuzumab) were incubated with cells and binding was determined using fluorophore-conjugated secondary anti-human IgG antibodies. Cells were analyzed by flow cytometry and the ratio of signal over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and trastuzumab normalized to control secondary antibodies.

HER2-TriNKET-A44, HER2-TriNKET-A49, and HER2-TriNKET-F63 showed comparable levels of binding to HER2 expressed on the surface of 786-O cells compared to trastuzumab (Fig. 8). HER2-TriNKET-E79 was shown to bind to HER2 expressed on the surface of NCI-H661 cells (Fig. 9).

Example 5 Trispecific binding proteins activate NK cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56+) were isolated using negative selection using magnetic beads from PBMCs, and the purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated NK cells were used within 24–48 h of activation.

Human cancer cells expressing tumor antigen were collected and resuspended in culture medium at 2x10 ⁶ cells/ml. Monoclonal antibodies or TriNKETs targeting tumor antigen were diluted in culture medium. Activated NK cells were collected, washed and resuspended at 2x10 ⁶ cells/ml in culture medium. Cancer cells were then mixed with monoclonal antibodies/TriNKET and NK cells were activated in the presence of IL-2. Brefeldin-A and monensin were also added to the mixed culture to block protein transport out of the cell for intracellular cytokine staining. Fluorophore-conjugated anti-CD107a antibody was added to the mixed culture and the culture was incubated for 4 hours before samples were prepared for FACS analysis using fluorophore-conjugated anti-CD3, CD56, and IFNgamma antibodies. CD107a and IFN-gamma staining was analyzed in CD3 ⁻ CD56 + cells to assess NK cell activation. The increase in CD107a/IFN-gamma double positive cells indicates better NK cell activation due to the involvement of two activating receptors rather than a single receptor.

- 30 043960

TriNKETs mediate activation of human NK cells co-cultured with

HE112-expressing NCI-H661 cells (Fig. 10) and SkBr-3 cells (Fig. 11), respectively, as shown by increased CD107a degranulation and IFN-gamma production. Compared to the monoclonal antibody trastuzumab, TriNKETs demonstrate superior activation of human NK cells in the presence of human cancer cells.

TriNKETs mediate activation of human NK cells co-cultured with CD33-expressing human AML Mv4-11 cells, as shown by increased CD107a degranulation and IFN-gamma production (Fig. 12). Compared to anti-CD33 monoclonal antibody, TriNKETs demonstrate superior activation of human NK cells in the presence of human cancer cells.

Example 6 Trispecific binding proteins result in cytotoxicity to target cancer cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of peripheral blood using density gradient centrifugation. NK cells (CD3-CD56+) were isolated using negative selection using magnetic beads from PBMCs, and the purity of isolated NK cells was typically >90%. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

To test the ability of human NK cells to lyse cancer cells in the presence of TriNKET, the cyto Tox 96 non-radioactive cytotoxicity assay from Promega (G1780) was used according to the manufacturer's instructions. Briefly, human cancer cells expressing tumor antigen were collected, washed and resuspended in culture medium at 1-2x105 cells/ml. Resting and/or activated NK cells were collected, washed and resuspended at 105-2.0x106 cells/ml in the same culture media as for cancer cells. In each well of a 96-well plate, 50 μl of a cancer cell suspension was mixed with 50 μl of an NK cell suspension with or without TriNKET targeting a tumor antigen expressed on cancer cells. After incubation at 37°C with 5% CO ₂ for 3 h and 15 min, 10x lysis buffer was added to wells containing only cancer cells and to wells containing only maximum lysis and negative control reagent media, respectively. The plate was then placed back into the incubator for an additional 45 min to achieve a total incubation of 4 h. The cells were then pelleted and the culture supernatant was transferred to a new 96-well plate and mixed with development substrate. The new plate was incubated for 30 min at room temperature and the absorbance was read at 492 nm on a SpectraMax i3x. The percentage of specific cancer cell lysis was calculated as follows:

% Specific lysis = ((experimental lysis - spontaneous lysis from NK cells only; spontaneous lysis from cancer cells only)/(Maximum lysis - negative reagent control)) x 100%.

TriNKETs mediate human NK cell cytotoxicity against the CD33-positive human AML cell line Molm-13. As shown in FIG. 13, resting human NK cells were mixed with Molm-13 cancer cells, and TriNKETs were able to enhance the cytotoxic activity of resting human NK cells in a dose-dependent manner against cancer cells. The dotted line indicates the cytotoxic activity of resting NK cells without TriNKET. As shown in FIG. 14, activated human NK cells were mixed with Molm-13 cancer cells, and TriNKETs increased the cytotoxic activity of activated human NK cells even more in a dose-dependent manner against cancer cells.

TriNKETs mediate human NK cell cytotoxicity against the HER2-positive human kidney cancer cell line 786-O. As shown in FIG. 15, resting human NK cells were mixed with 786-O cancer cells, and TriNKETs were able to enhance the cytotoxic activity of resting human NK cells in a dose-dependent manner against cancer cells (each TriNKET was added at a concentration of 5, 1, 0.2 μg/ml in analysis, and the results are presented in three columns from left to right in each TriNKET in Fig. 15, 16). The dotted line indicates the cytotoxic activity of resting NK cells against 786-O cells in the absence of TriNKET. As shown in FIG. 16, activated human NK cells were mixed with 786-O cancer cells, and TriNKET increased the cytotoxic activity of activated human NK cells even more in a dose-dependent manner against cancer cells. The dotted line indicates the cytotoxic activity of activated NK cells against 786-O cells in the absence of TriNKET.

Example 7: ADI-27749 and TriNKET variants containing variants.

As described above, ADI-27749 (A49) contains, among other things, a CDR3 heavy chain having the amino acid sequence GAPMGAAAGWFDP (SEQ ID NO: 71). Met at position 102 of SEQ ID NO: 7 (i.e. at position 4 of this CDR3 sequence) can be replaced by Gln, Leu, Ile, Phe or Val, thereby generating antibodies to NKG2D A49MQ, A49ML, A49MI, A49MF and A49MV respectively, having a corresponding heavy chain variable region, variable region

- 31 043960 light chain and CDR sequences presented in table. 1.

The effects of these mutations on hydrophobicity were analyzed using MOE2018.01 using the parameters avg_pro_patch_cdr_hyd. Residues were mutated using the protein builder module, and the entire Fab was minimized after all residues were attached. Dynamic feature sampling was performed using the lowMD protocol in BIOMOE. As shown in table. 11, these mutations did not have a significant negative effect on the predicted hydrophobicity of A49 Fab.

_____________________________________________________Table 11

Amino acid residue avg_pro_patch_cdr_hyd M 524.0968 L 529.67743 I 551.93549 V 477.09677 Q 447.09677 F 542.25806

The hydrophobicity of TriNKET containing A49 (TriNKET A) and a mutant form of TriNKET A substituting Ile, Leu, Val, Gln or Phe for Met (TriNKET A*) were tested by analytical hydrophobic interaction chromatography (HIC). Each of the TriNKETs is also associated with a first tumor antigen. As shown in table. 12, the retention time of TriNKET A* was the same as that of TriNKET A.

Table 12

Protein Hold Time ΪΤ A* 8.6 min TriNKET A 8.65 ± 0.05 min

The thermal stability of TriNKET A and TriNKET A* was studied using differential scanning calorimetry (DSC) analysis in 20 mM histidine, 260 mM sucrose and 0.005% PS-80 at pH 6.0. Tm values are given in table. 13, where Tm represents the midpoint of the transition temperature of an individual domain. The M102 mutation had a small effect on the Tm value of the two most stable transitions ( _Tm3 and _Tm4 ), with a shift of 0.6 and 0.7°C lower compared to TriNKET A. The earlier transitions ( _Tm1 and Tm2) were not affected. Therefore, the M102 mutation had only a minor effect on the overall thermostability of TriNKET A.

Table 13

Protein T _t 1 T _t 2 t _t z T _t 4 TriNKET A 66.2 80.2 86.3 88.4 TriNKET A* 66.2 80.5 85.7 87.7

Binding of TriNKET A and TriNKET A* to human NKG2D-mouse Fc fusion protein (mFc-hNKG2D) was characterized by surface plasmon resonance (SPR) at 37°C. Two different fits were used to obtain the equilibrium affinity data: the steady state affinity fit and the kinetic affinity fit (FIG. 32). Kinetic constants and equilibrium affinity constants were calculated, and data from two independent experiments for TriNKET A* and three independent experiments for TriNKET A were averaged.

Table 14

Capture Analyst K(1/Mc) Ml/c) Kinetics ^d(M) Equilibrium affinity ^d(M) Stoichiomet Riya mFc- hNKG2D TriNKET A* 1.41 x 10 ⁵ 1.31 X 10 ¹ 9.31 x IO' ⁷ 6.98 x ^IO'7 0.86 mFc- hNKG2D TriNKET A* 1.56 x 10 ⁵ 1.28 x 10' ¹ 8.19 x IO' ⁷ 6.76 x ^IO'7 0.85 Average 1.49 x 10 ⁵ 1.30 X 10 ¹ 8.75 x IO' ⁷ 6.87 x ^IO'7 0.85 mFc- hNKG2D TriNKET A 1.91 x 10 ⁵ 1.16 X 10 ¹ 6.05 x IO' ⁷ 4.62 x ^IO'7 1.01 mFc- hNKG2D TriNKET A 2.03 x 10 ⁵ 1.06 X 10 ¹ 5.23 x IO' ⁷ 4.20 x IO' ⁷ 0.88 mFc- hNKG2D TriNKET A 1.93 x 10 ⁵ 1.15 x 10' ¹ 5.95 x IO' ⁷ 5.80 x IO' ⁷ 1.12 Mean ± STDEV (1.96 ± 0.06) x 10 ⁵ (1.12 ±0.06) x 10 ¹ (5.74 ± 0.45) x IO' ⁷ (4.87 ± 0.83) x IO' ⁷ 1.01 ± 0.11

- 32 043960

As shown in table. 14, the equilibrium affinity constants (KD) obtained from the affinity and kinetics fits were very similar between independent experiments, indicating high reliability of the measured parameters. The M102 variant has less than a 2-fold reduction in affinity for human NKG2D compared to TriNKET A. KD TriNKET A* was (6.87±0.16)x 10 ^-7 M, while KD TriNKET A was (4 .87±0.83)x10 ^-7 M (calculated from affinity fitting). Similar differences in affinity were observed when KD was calculated using kinetics fitting. The binding stoichiometry of NKG2D to TriNKET A* was 0.85 ± 0.12, similar to 1.01 ± 0.11 for TriNKET A, confirming that each NKG2D dimer binds to one TriNKET A* molecule. This suggests that the M102 mutation had only a minor effect on the binding of A49-containing TriNKET to human NKG2D.

Finally, the effect of the M102 mutation on TriNKET activity was assessed in a cytotoxicity assay. Briefly, KHYG-1 cells expressing a high-affinity variant of CD16a (158 B) were generated by retroviral transduction. Following transduction, cells were selected in growth medium containing puromycin to generate a selected population of KHYG-1-CD16V cells. The selected population was maintained in medium containing 10 ng/ml human IL-2. To prepare KHYG-1-CD16V cells for use as effectors in cytotoxicity assays, cells were harvested from culture, pelleted, washed three times in culture medium without IL-2, and resuspended in culture medium without IL-2 and left for 24 h.

Human cancer cell lines expressing the target of interest were collected from culture. Cells were washed with HBS and resuspended in growth medium at 106 cells/ml for labeling with BATDA reagent (Perkin Elmer C136-100). The manufacturer's instructions for labeling target cells were followed. After labeling, cells were washed three times with HBS and resuspended at 0.5 x 10 ⁵ cells/ml in culture medium. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate.

TriNKET was serially diluted in culture medium, and 50 μl of diluted TriNKET was added to each well. Resting NK cells were collected from culture, washed and resuspended at 1.0 x 10 ⁶ cells/ml in culture medium. 50 μl of NK cells was added to each well of the plate to achieve the desired E:T ratio of 10:1 and to obtain a total culture volume of 200 μl in each well. The plate was incubated at 37°C with 5% CO ₂ for 2-3 hours.

After cultivation, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200xg for 5 min. 20 μL of culture supernatant was transferred into a clean microplate provided by the manufacturer. Supernatant from labeled cells incubated separately without NK cells was used to measure spontaneous TDA release. Supernatant from labeled cells incubated with 1% Triton-X was used to measure maximum lysis of target cells. The supernatant from labeled cells 2–3 h before incubation was used for background measurements and for quality control purposes.

200 μl of room temperature europium solution (Perkin Elmer C135-100) was added to each well containing the culture supernatant. The plate was protected from light and incubated on a shaker at 250 rpm for 15 min. Fluorescence was measured using a SpectraMax i3X instrument. Fluorescence levels represented lysis of target cells. % Specific lysis was calculated as:

% Specific Lysis = ((Experimental Release - Spontaneous Release)/(Maximum Release - Spontaneous Release)) x 100%.

To measure the activity of TriNKET A and TriNKET A*, a cell line that expressed the first tumor antigen was selected as target cells. Two different batches of TriNKET A were used for comparison. The % Specific Lysis values were plotted in FIG. 33, and the EC ₅₀ values and the maximum % specific lysis are summarized in table. 15. The EC ₅₀ values and maximum % specific lysis of TriNKET A* were similar to those of TriNKET A, suggesting that the M102 mutation did not affect the biological activity of TriNKET A.

Table 15

Protein EC ₅₀ (nM) Maximum lysis (%) TriNKET A* 0.15 73 TriNKET A - batch 1 0.17 76 TriNKET A - batch 2 0.15 76

To confirm that the lack of effect of the M102 mutation on TriNKET activity was not tumor antigen specific, TriNKET A and TriNKET A* were designed to bind to a second, separate tumor antigen. The activities of the two TriNKETs were compared in cytotoxicity assays using a cell line that expressed the second tumor antigen as target cells and KHYG-1-CD16V cells as effector cells. As shown in FIG. 34, TriNKET A* demonstrated activity equivalent to TriNKET A.

-

Claims (37)

Incorporation by reference The entire disclosure of each of the patent documents and scientific articles mentioned herein is incorporated by reference for all purposes. Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Thus, the above embodiments should be considered in all respects as illustrative and not limiting of the invention described herein. Accordingly, the scope of the present invention is indicated by the appended claims and not by the preceding description, and all modifications that come within the meaning and scope of the claims are intended to be included therein. CLAIM 1. Antigen-binding site that binds to the natural killer group 2D receptor (NKG2D), containing: a heavy chain variable domain comprising a complementarity determining region 1 (CDR1) sequence with the amino acid sequence SEQ ID NO: 48, a complementarity determining region 2 (CDR2) sequence with the amino acid sequence SEQ ID NO: 30, and a complementarity determining region 3 (CDR3) sequence with the amino acid sequence SEQ ID NO: 44; and a light chain variable domain comprising a CDR1 sequence having the amino acid sequence SEQ ID NO: 32, a CDR2 sequence having the amino acid sequence SEQ ID NO: 33, and a CDR3 sequence having the amino acid sequence SEQ ID NO: 34. 2. The antigen-binding region according to claim 1, where the heavy chain variable domain contains: CDR1 with amino acid sequence SEQ ID NO: 29; CDR2 with amino acid sequence SEQ ID NO: 30 and CDR3 with amino acid sequence SEQ ID NO: 31. 3. The antigen-binding region according to claim 1, where the heavy chain variable domain contains: CDR1 with amino acid sequence SEQ ID NO: 48; CDR2 with amino acid sequence SEQ ID NO: 30 and CDR3 with amino acid sequence SEQ ID NO: 71. 4. The antigen-binding region according to claim 1, where the heavy chain variable domain contains: CDR1 with amino acid sequence SEQ ID NO: 29; CDR2 with amino acid sequence SEQ ID NO: 30 and CDR3 with amino acid sequence SEQ ID NO: 77. 5. The antigen-binding region according to claim 1, where the heavy chain variable domain contains: CDR1 with amino acid sequence SEQ ID NO: 48; CDR2 with amino acid sequence SEQ ID NO: 30 and CDR3 with amino acid sequence SEQ ID NO: 78. 6. Antigen-binding site according to any one of claims 1 to 5, where: the antibody heavy chain variable domain contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7; and the antibody light chain variable domain contains an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 8. 7. The antigen binding site according to any one of claims 1 to 6, wherein the antigen binding site binds to NKG2D with a KD between 2-120 nM, as measured by surface plasmon resonance. 8. A protein that binds to NKG2D and tumor-associated antigen, where the protein contains: an antigen binding region that binds to NKG2D according to any one of claims 1 to 7 and an additional antigen binding region that binds to a tumor-associated antigen, wherein the additional antigen binding region comprises: (i) a heavy chain variable domain and a light chain variable domain or (ii) a single domain antibody. 9. The protein according to claim 8, where the tumor-associated antigen is selected from the group consisting of HER2, EpCAM, CD2, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4 , MUC1, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1. 10. Protein according to claim 8 or 9, where: antigen binding region heavy chain variable domain that binds NKG2D, - 34 043960 is present on the first polypeptide, additionally containing the first constant region of the antibody; and an additional antigen binding region heavy chain variable domain that binds to a tumor-associated antigen is present on the second polypeptide further comprising a second antibody constant region. 11. The protein of claim 10, wherein the first antibody constant region and the second antibody constant region form a complex capable of binding CD 16. 12. The protein of claim 10 or 11, wherein each of the first antibody constant region and the second antibody constant region comprises a hinge, CH2 and CH3 domains, and optionally CH1 domains. 13. The protein according to any one of claims 10 to 12, wherein the amino acid sequences of each of the first antibody constant region and the second antibody constant region are at least 90% identical to the human IgG1 constant region. 14. Protein according to any one of claims 10-13, where: the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region at positions Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405 , Y407, K409, T411 or K439 or any combination thereof; the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region at position Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409 , T411 or K439 or any combination thereof. 15. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and where the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, L368 or Y407, or any combination thereof. 16. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366, L368 or Y407 or any combination thereof and wherein the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the constant region IgG1 region at position T366. 17. The protein according to any one of claims 10-13, where the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region at position E357, K360, Q362, S364, L368, K370, T394, D401, F405 or T411, or any of them combinations and wherein the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at position Y349, E357, S364, L368, K370, T394, D401, F405 or T411 or any combination thereof. 18. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region at position Y349, E357, S364, L368, K370, T394, D401, F405 or T411 or any combination thereof and wherein the amino acid sequence of the second antibody constant region differs from the amino acid sequence of the IgG1 constant region at position E357, K360, Q362, S364, L368, K370, T394, D401, F405 or T411 or any combination thereof. 19. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position L351, D399, S400 or Y407 or any combination thereof and wherein the amino acid sequence of the second antibody constant region is different from the amino acid sequence IgG1 constant region sequences at position T366, N390, K392, K409 or T411 or any combination thereof. 20. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position T366, N390, K392, K409 or T411 or any combination thereof and wherein the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region at position L351, D399, S400 or Y407 or any combination thereof. 21. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position Q347, Y349, K360 or K409 or any combination thereof and wherein the amino acid sequence of the second antibody constant region is different from the amino acid sequence IgG1 constant region sequences at position Q347, E357, D399 or F405 or any combination thereof. 22. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first constant region of the antibody is different from the amino acid sequence of the IgG1 constant region at position Q347, E357, D399 or F405 or any combination thereof and where the amino acid sequence of the second constant region of the antibody is different from the amino acid sequence constant sequence - 35 043960 IgG1 region at position Y349, K360, Q347 or K409 or any combination thereof. 23. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position K370, K392, K409 or K439 or any combination thereof and where the amino acid sequence of the second antibody constant region is different from the amino acid sequence IgG1 constant region sequence at position D356, E357 or D399 or any combination thereof. 24. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first constant region of the antibody is different from the amino acid sequence of the constant region of IgG1 at position D356, E357 or D399 or any combination thereof and where the amino acid sequence of the second constant region of the antibody is different from the amino acid sequence of the constant region the IgG1 region at position K370, K392, K409 or K439 or any combination thereof. 25. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position L351, E356, T366 or D399 or any combination thereof and where the amino acid sequence of the second antibody constant region is different from the amino acid sequence IgG1 constant region sequences at position Y349, L351, L368, K392 or K409 or any combination thereof. 26. The protein according to any one of claims 10 to 13, wherein the amino acid sequence of the first antibody constant region is different from the amino acid sequence of the IgG1 constant region at position Y349, L351, L368, K392 or K409 or any combination thereof and wherein the amino acid sequence of the second antibody constant region is different from the amino acid sequence of the IgG1 constant region at position L351, E356, T366 or D399 or any combination thereof. 27. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution S354C and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by the substitution Y349C. 28. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution Y349C, and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by the substitution S354C. 29. The protein according to any one of claims 10-13, where the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions K36OE and K409W and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions O347R, D399V and F405T. 30. The protein according to any one of claims 10-13, where the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions O347R, D399V and F405T and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions K36OE and K409W. 31. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first antibody constant region differs from the amino acid sequence of the IgG1 constant region by the substitution T366W and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by the substitutions T366S, T368A and Y407V. 32. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions T366S, T368A and Y407V and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitution T366W. 33. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions T350V, L351Y, F405A and Y407V and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions T350V, T366L, K392L and T394W. 34. The protein according to any one of claims 10-13, wherein the amino acid sequence of the first constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions T350V, T366L, K392L and T394W and where the amino acid sequence of the second constant region of the antibody differs from the amino acid sequence of the IgG1 constant region by substitutions T350V, L351Y, F405A and Y407V. 35. The protein according to any one of claims 8 or 9, wherein the protein further comprises an antigen-binding site capable of binding CD16. 36. A composition for the treatment of cancer, comprising a protein according to any one of claims 8 to 35 and a pharmaceutically acceptable carrier. 37. A host cell for producing a protein containing one or more nucleic acids encoding -