Twin immune cell engager

Abstract

To provide kits or compositions for treating cancer.SOLUTION: The invention provides a kit or composition for treating cancer comprising a. a first component comprising a targeted immune cell binding agent comprising: i. a first targeting moiety that binds to a tumor antigen expressed by the cancer; ii. a first immune cell binding domain being either a VH domain or VL domain, that can exert immune cell binding activity in association with a second immune cell binding domain not part of the first component; and iii. a first complementary binding domain capable of binding to a complementary antigen in association with a second complementary domain; b. a second component comprising a targeted immune cell binding agent that comprises: i. a second targeting moiety, ii. a second complementary binding domain capable of exerting immune cell biding activity in association with the first immune cell binding domain.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 780,770, filed December 17, 2018. This application claims the benefit of priority to the present application, which is hereby incorporated by reference in its entirety for all purposes. Reference is made to the detailed text.

[0002] Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing is 212,992 bytes in size. Created on December 12, 2019 by 2019-12-12_01131-002 The file is provided as a file entitled "3-00PCT_ST25.txt". The information in the sequence listing is incorporated herein by reference in its entirety.

[0003] Field The present application relates to improved targeted immunotherapy for treating disease states characterized by the presence of cancer cells. The present invention relates to an immune cell-engaging antibody complex that inhibits a disease state characterized by the presence of cancer cells. It relates to drugs that can be used to treat [Background technology]

[0004] Cancer causes significant loss of life, suffering and economic devastation. Immunotherapeutic strategies have become an active area of ​​clinical translational research.

[0005] A variety of immunotherapy treatments to combat cancer and other diseases caused by the presence of unwanted cells. Oncological approaches have been developed. In some circumstances, targeted T cell-engaging antibody complexes are useful. These masking methods provide promising data. See US 10,035,856. The labeled composition includes a first component and a second component, which bind to the unwanted cells. and releasing inactive binding partners from each component in the tumor microenvironment. Each is then available for combination with the other. After binding to the T-cell receptor, they form an active T-cell binding domain.

[0006] Other advances include the potential for other immune interventions (in addition to activating T cells) to treat cancer and other diseases. For example, PD-L1 pathway inhibition has been shown to be beneficial for patients with and can reverse T cell exhaustion in some tumors (Chen DS e t al.Clin Cancer Res.18:6580-6587(2012) Activation of T cells and binding of tumor-associated antigens, such as checkpoint molecules Simultaneous use of these two drugs may provide a more robust and selective strategy for activating T cells against cancer cells. This has been proposed as a useful method (Harris et al. Cancer Biol M ed 13(2):171-193(2016), and Kobold et al. Ont Oncol 8:285(2018). However, various A treatment approach that incorporates all of these features so that function is active only at the desired treatment site is Not one.

[0007] Thus, depending on the disease and patient, additional immuno-oncology or other biological interventions may be warranted. Some conventional constructs may be able to activate T cells in the tumor microenvironment, but these may also favor tumor-associated tumors. It had the ability to activate the nemesis, but had no other attributes. Conventional constructs restricted to the desired therapeutic site offer the opportunity to incorporate additional functional domains. Therefore, further development in the field of targeting immunotherapy has been achieved. Further development is required. Summary of the Invention

[0008] The TWICE complex of the present invention is a two-component complex that activates multiple functions in the tumor microenvironment. Thus, the complexes of the present invention provide a unique ability to integrate into the tumor microenvironment. The two-component complex has the ability to be localized and induce two distinct signals to benefit patients. This unique construct provides a significant advantage in having a single approach to offers advantages not available in prior art constructions.

[0009] The application is described as a kit or composition for treating cancer in a patient. a first component comprising a targeted immune cell binding agent, The combination comprises a first targeting moiety that binds to a tumor antigen expressed by the cancer; immune cell binding activity when bound to a second immune cell binding domain that is not part of the The first immune cell binding domain is either a VH domain or a VL domain that can bind to the first immune cell. and binding to a complementary antigen when bound to a second complementary binding domain. and a second complementary binding domain capable of binding to the first and when the first immune cell binding domain is a VH domain, the complementary binding domain is a VL domain and the first immune cell binding domain is a VL domain and the first complementary binding domain is a VH domain when The first immune cell-binding domain binds to the first complementary domain of the first immune cell. The first immune cell-binding domain is not capable of binding to the second immune cell-binding domain. The first component, which is a binding partner of the cell-binding domain, and a targeting immune a second component comprising a cell binding agent, the targeted immune cell binding agent binding to a second targeting molecule; a tropic portion that exhibits an immune cell binding effect when bound to the first immune cell binding domain; and when the first immune cell binding domain is VL, it is VH, and the first immune cell binding domain is VL. a second immune cell binding domain that is a VL when the domain is a VH, and a first complementary A second complementary binding domain capable of binding to a complementary antigen when bound to the complementary binding domain. and when the second immune cell binding domain is a VH domain, the second immune cell binding domain is The complementary binding domain of the first immune cell is a VL domain and the second immune cell binding domain is a VL domain. and the second complementary binding domain is a VH domain when the second complementary binding domain is a The second immune cell binding domain is bound to the second complementary domain. A second immune cell binding domain that does not bind to the first immune cell binding domain. said kit comprising said second component which is a binding partner of said immune cell binding domain, or The composition will be described.

[0010] In some embodiments, the first immune cell binding domain comprises a first dimerization domain and The first dimer is bound to the first complementary binding domain by the second dimerization domain. The somatization domain is linked to the first immune cell binding domain by a first linker, The second dimerization domain is linked to the first complementary binding domain by a second linker. and wherein the first and / or second linker is a cleavable linker. wherein the first and second linkers are cleavable linkers.

[0011] In some embodiments, the second T cell binding domain comprises a first dimerization domain and The first dimer is bound to a second complementary binding domain by a dimerization domain of 2. The immunization domain is linked to a second T cell binding domain by a first linker, the dimerization domain is linked to a second complementary binding domain by a second linker; The first and / or second linker is a cleavable linker.

[0012] In some embodiments, the first and second linkers are cleavable linkers. In some embodiments, the first linker and the second linker are the same. In some embodiments, the first linker and the second linker are different. The length of the first and second linkers is 5 to 30 amino acids. The first and second linkers are 8 to 16 amino acids in length. and / or the protease cleavage site of the second cleavable linker is a cancer or tumor microcirculation In some embodiments, the cleavage occurs due to a protease expressed by the host cell. The protease cleavage site of the first and / or second cleavable linker is located in the first component or The targeting moiety in at least one of the two components may be the same or different and may be targeted to cancer. Thus, a targeting moiety that is an antibody or antigen-binding fragment thereof that binds to an expressed tumor antigen It is cleaved by proteases that are co-localized in tumors.

[0013] In some embodiments, the first and second dimerization domains are both leucine nucleotides. A domain can be a peptide, an immunoglobulin domain, or a T cell receptor (TCR) domain. In some embodiments, the immunoglobulin domain is an immunoglobulin variable domain or an immunoglobulin variable domain. In some embodiments, the immunoglobulin constant domain comprises an immunoglobulin constant domain. , CH1 / CL, CH2, CH3 or CH4. In some embodiments, the TCR In some embodiments, the domain comprises a TCR constant domain. The dimerization domain is the same as the dimerization domain in the second component. In this case, the dimerization domain in the first component is different from the dimerization domain in the second component. It has become.

[0014] In some embodiments, the first complementary binding domain and the second complementary binding domain are and when bound to each other, are capable of binding to the cancer. The first complementary binding domain and the second complementary binding domain form an immunosuppressant when bound to each other. Binds to immune checkpoint molecules, RANK or RANKL, or cell death-inducing antigens It is possible.

[0015] In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound to each other, they can bind to immune checkpoint molecules. In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound, it is capable of binding to PD-L1. In some embodiments, the first and the second complementary binding domain is atezolizumab, durvalumab or avelumab. Includes all or part of VH and / or VL.

[0016] In some embodiments, the first complementary binding domain and the second complementary binding domain are , when bound to each other, can bind to RANK. When the first complementary binding domain and the second complementary binding domain are bound to each other, In some embodiments, the first and second complementary The binding domain comprises all or a portion of the VH and / or VL of denosumab.

[0017] In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound to each other, they can bind to a cell death-inducing antigen. In this embodiment, the first complementary binding domain and the second complementary binding domain are bound to each other. When Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, and DR3 / A po3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Ap Binds to o2 / TRAIL-R2 / TRICK2 / KILLER, DR6, or CAR1 In some embodiments, the first complementary binding domain and the second complementary binding domain can be The specific binding domains bind to TRAIL-R1 / DR4 when bound to each other. In some embodiments, the first and second complementary binding domains are In some embodiments, the first phase comprises all or a portion of the VH and / or VL of The complementary binding domain and the second complementary binding domain bind to DR5 / It can bind to Apo2 / TRAIL-R2 / TRICK2 / KILLER. In some embodiments, the first and second complementary binding domains are conatumumab (AMG6 55), lexatumumab, tigatuzumab (CS1008) or drozitumab (PRO9 5780), including all or part of the VH and / or VL.

[0018] In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound to each other, they can bind to molecules associated with the extracellular matrix. do.

[0019] In some embodiments, the first complementary binding domain and the second complementary binding domain are , when bound to each other, bind to T cells, macrophages, or natural killer cells It is possible.

[0020] In some embodiments, the first complementary binding domain and the second complementary binding domain are and can bind to T cells when bound to each other. In some embodiments, The first complementary binding domain and the second complementary binding domain, when bound to each other, CD3, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein CTLA-4, T cell immunoglobulin mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD28, CD137, OX40, CD27, glucocorticoid-induced TNF receptor (G ITR or TNFRSF18), a T cell immunoreceptor with Ig and ITIM domains (TIGIT), or inducible T cell costimulator (ICOS).

[0021] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary binding sites are capable of binding to CD3. The combined domains are muromonab, otelixizumab, teplizumab, visilizumab, and foral. It contains all or part of the VH and / or VL of mab, SP34 or blinatumomab.

[0022] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The binding domain may be the entire or partial VH and / or VL of pembrolizumab or nivolumab. includes some.

[0023] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second phase The complementary binding domain comprises all or a portion of the VH and / or VL of ipilimumab.

[0024] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The specific binding domain may be the entire VH and / or VL of TSR-022 or Sym023. or a part thereof.

[0025] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The specific binding domain comprises all or a portion of the VH and / or VL of BMS-986016. nothing.

[0026] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary binding sites are capable of binding to KIR. The binding domain comprises all or a portion of the VH and / or VL of lirilumab.

[0027] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The binding domain comprises all or a portion of the VH and / or VL of ceralizumab.

[0028] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The specific binding domain is the entire VH and / or VL of utomirumab or urelumab, or Includes some.

[0029] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The binding domain is the VH and / or VH of PF-04518600 or BMS986178. Contains all or part of the VL.

[0030] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The binding domain comprises all or a portion of the VH and / or VL of varlilumab.

[0031] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the IL-16 polypeptide can bind to GITR (TNFRSF18). The first and second complementary binding domains are the VH of GWN323 or BMS-986156. and / or includes all or part of VL.

[0032] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The specific binding domains are OMP-313M32, MTIG7192A, and BMS-986207. or comprising the whole or part of the VH and / or VL of MK-7684.

[0033] In some embodiments, the first and second complementary binding domains are associated with each other. In some embodiments, the first and second complementary The binding domain comprises all or part of the VH and / or VL of JTX-2011.

[0034] In some embodiments, the first complementary binding domain and the second complementary binding domain are , and when bound to each other, can bind to macrophages.

[0035] In some embodiments, the first complementary binding domain and the second complementary binding domain are and can bind to CSF1R when bound to each other. The first and second complementary binding domains are VH of emactuzumab or IMC-CS4. and / or includes all or part of VL.

[0036] In some embodiments, the first complementary binding domain and the second complementary binding domain are and are capable of binding to CD40 when bound to each other. The first and second complementary binding domains are the VH and / or VL of CP-870,893. Includes all or part of.

[0037] In some embodiments, the first complementary binding domain and the second complementary binding domain are , and when bound to each other, are capable of binding to natural killer cells.

[0038] In some embodiments, the first complementary binding domain and the second complementary binding domain are and are capable of binding to CD16A when bound to each other. The first and second complementary binding domains are VH and VH of NTM-1633 or AFM13. and / or VL.

[0039] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains are capable of binding to the same antigen.

[0040] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains can bind to different antigens on the same cell.

[0041] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains are capable of binding to different cells.

[0042] In some embodiments, the first and second immune cell binding domains are first and second phase Fv can be formed when not bound to a complementary binding domain. In one embodiment, the first and second complementary binding domains are first and second immune cell binding domains. When not bound to the Fv, it can form an Fv.

[0043] The present application also provides a kit or composition for treating cancer in a patient, comprising: a first component comprising a targeted immune cell binding agent, the targeted immune cell binding agent comprising A first targeting moiety that binds to a tumor antigen expressed by the cancer, the first targeting moiety being not part of the first component. and having an immune cell-binding effect when bound to a second immune cell-binding domain that is a first immune cell binding domain, which may be either a VH domain or a VL domain, a first inactive binding partner of the first immune cell binding domain, Unless the toner is removed, the first immune cell binding domain binds to the second immune cell binding domain. The first immune cell-binding domain binds to the first immune cell-binding domain but does not bind to the second immune cell-binding domain. When the first immune cell binding domain is a VH domain, the second immune cell binding domain is a VL domain. When the cell-binding domain is a VL domain, the first inactive binding domain is a VH domain. The binding partner separates the first immune cell binding domain from the first inactive binding partner. a protease cleavage site expressed by cancer or tumor microenvironment cells; or (a) a first and / or second targeting moiety in the agent that is the same or different. or (b) cells in the tumor microenvironment that bind to tumor antigens expressed by the cancer. The targeting moiety is an antibody or an antigen-binding fragment thereof that binds to an antigen expressed by Thus, in the presence of proteases that are colocalized in tumors, immune cell-binding domains the protease cleavage site capable of releasing the inactive binding partner from the and a first complementary functional domain capable of immune cell binding. and a second component comprising a targeted immune cell binding agent, The cell binding agent comprises a second targeting moiety, a second immune cell binding domain, and optionally a second component comprising a second complementary functional domain capable of immune cell binding; Also described are kits or compositions comprising the same.

[0044] In some embodiments, the second component comprises a supplemental functional domain. In one embodiment, the complementary functional domain of the first and / or second component comprises a ligand for a receptor. In some embodiments, the supplemental functional domains are members of the TGF-beta family. In some embodiments, the first and / or second components may be supplemented with In some embodiments, the target functional domain comprises a cytokine. L-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or a member of the TNF superfamily.

[0045] In some embodiments, the complementary functional domains of the first and / or second components are attenuated. In some embodiments, the attenuated cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-22, IL- L-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ or TNF It is a variant of a member of the superfamily.

[0046] In some embodiments, the second component comprises a second inactive linker of a second immune cell binding domain. The second inactive binding partner is further included in the inactive binding partner. The second immune cell-binding domain will not bind to the first immune cell-binding domain unless the second immune cell-binding domain is removed. The second immune cell-binding domain binds to the second immune cell, so that the second immune cell does not bind to the second immune cell. When the cell-binding domain is a VH domain, the inactive binding partner is a VL domain. and the second immune cell binding domain is a VL domain and the inactive binding partner is a VH domain, and further comprising a protease cleavage site between the second immune cell binding domain and the second immune cell binding domain. The protease cleavage site separates the nuclease from its inactive binding partner, which is expressed by cancer. or (a) the same as the first and / or second targeting moieties in the agent. or (b) the tumor microenvironment, which binds to tumor antigens expressed by the cancer and are distinct from the tumor A targeting antibody or antigen-binding fragment thereof that binds to an antigen expressed by a cell in a subject. Immune cell binding in the presence of proteases that are colocalized to tumors by their functional moieties. The inactive binding partner can be released from the binding domain.

[0047] In some embodiments, the first component is not covalently bonded to the second component. In some embodiments, the first moiety is covalently bonded to the second moiety. In embodiments, the first component is covalently attached to the second component via a cleavable linker. do.

[0048] In some embodiments, the first immune cell binding domain and the second immune cell binding domain When bound to each other, the antibodies bind to T cells, macrophages, or natural killer cells. can be combined.

[0049] In some embodiments, the first immune cell binding domain and the second immune cell binding domain In some embodiments, the antibodies can bind to T cells when bound to each other. The first immune cell binding domain and the second immune cell binding domain are bound to each other. CD3, T cell receptor, programmed cell death protein 1 (PD-1), and cytotoxicity Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin mucin domain TIM-3, lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin KIR, CD28, CD137, OX40, CD27, GITR (TNF RSF18), TIGIT or inducible T cell costimulatory factor (ICOS). can.

[0050] In some embodiments, the first immune cell binding domain and the second immune cell binding domain In some embodiments, the agonists are capable of binding to CD3 when bound to each other. The first and second immune cell binding domains are muromonab, otelixizumab, teplizumab, VH of mab, visilizumab, foralumab, SP34 or blinatumomab and / or Contains all or part of the VL.

[0051] In some embodiments, the first immune cell binding domain and the second immune cell binding domain The antibodies can bind to the T cell receptor when bound to each other.

[0052] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune cells are capable of binding to PD-1. The cell-binding domain is the entire VH and / or VL of pembrolizumab or nivolumab. Or part of it.

[0053] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second The immune cell binding domain comprises all or a portion of the VH and / or VL of ipilimumab. .

[0054] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immunoglobulins are capable of binding to TIM-3. The immune cell binding domain is the entire VH and / or VL of TSR-022 or Sym023. Includes the body or part.

[0055] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immunoglobulins are capable of binding to LAG-3. The immune cell binding domain is the whole or a part of the VH and / or VL of BMS-986016. Includes.

[0056] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune cells are capable of binding to a KIR. The cell-binding domain comprises all or a portion of the VH and / or VL of lirilumab.

[0057] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune cells are capable of binding to CD28. The cell-binding domain comprises all or a portion of the VH and / or VL of ceralizumab.

[0058] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immunoglobulins are capable of binding to CD137. The immune cell-binding domain may be the entire VH and / or VL of utomirumab or urelumab. or a part thereof.

[0059] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune responses can bind to OX40. The cell-binding domain is the VH and / or VL of PF-04518600 or BMS986178. or VL.

[0060] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune cells are capable of binding to CD27. The cell-binding domain comprises all or a portion of the VH and / or VL of varlilumab.

[0061] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the antibody can bind to GITR (TNFRSF18). The first and second complementary binding domains are V of GWN323 or BMS-986156. Includes all or part of H and / or VL.

[0062] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune The immune cell binding domains are OMP-313M32, MTIG7192A, and BMS-9862. It contains all or part of the VH and / or VL of 07 or MK-7684.

[0063] In some embodiments, the first and second immune cell binding domains are linked together. In some embodiments, the first and second immune cells can bind to ICOS. The cell-binding domain comprises all or part of the VH and / or VL of JTX-2011. .

[0064] In some embodiments, the first immune cell binding domain and the second immune cell binding domain The antibodies can bind to macrophages when bound to each other.

[0065] In some embodiments, the first immune cell binding domain and the second immune cell binding domain The agonists are capable of binding to CSF1R when bound to each other. In this embodiment, the first and second immune cell binding domains are selected from the group consisting of emactuzumab and IMC-CS4. It comprises all or part of the VH and / or VL of the above.

[0066] In some embodiments, the first immune cell binding domain and the second immune cell binding domain The antibodies can bind to CD40 when bound to each other. In the above, the first and second immune cell binding domains are the VH and / or VH of CP-870,893. comprises all or part of VL.

[0067] In some embodiments, the first immune cell binding domain and the second immune cell binding domain When bound to each other, the agonists can bind to natural killer cells. In some embodiments, the first immune cell binding domain and the second immune binding domain are When bound, it is capable of binding to CD16A.

[0068] In some embodiments, the first and second immune cell binding domains are NTM-1633 or the whole or part of the VH and / or VL of AFM13. In the above, the first targeting moiety and the second targeting moiety are different.

[0069] In some embodiments, the first targeting moiety and the second targeting moiety are the same. do.

[0070] In some embodiments, the first and / or second targeting moiety is an antibody or In some embodiments, the first and / or second targeting moiety comprises an antigen-binding fragment. The study included DNA aptamers, RNA aptamers, albumin, lipocalin, fibronectin, and Ankyrin, Finomer, Obody, DARPin, knotin , avimer, atrimer, anticalin, affilin, aff antibody, bicyclic peptide, cys-knot, FN3 (adnectin, centrilin ( centryrins), pronectin or TN3), or Kunitz-type domains Includes.

[0071] In some embodiments, the second targeting moiety targets a tumor antigen expressed by the cancer. In some embodiments, the first and / or second targeting moiety binds to an α4 antibody. Integrin, A33, ACVRL1 / ALK1, ADAM17, ALK, APRIL, B CMA, C242, CA125, cadherin-19, CAIX, CanAg, carbonic anhydrase Element IX, CCN1, CCR4, CD123, CD133, CD137(4-1BB), C D138 / syndecan-1, CD19, CD2, CD20, CD22, CD30, CD33 , CD37, CD38, CD4, CD40, CD44, CD45, CD48, CD5, C D52, CD56, CD59, CD70, CD70b, CD71, CD74, CD79b , CD80, CD86, CD98, CEA, CEACAM, CEACAM1, CK8, c -Kit, Claudin-1 (CLDN1), CLDN18 (including CLDN18.2) , CLDN6, c-met / HGFR, c-RET, Cripto, CTLA-4, CX CR4, DKK-1, DLL3, DLL4, TRAIL-R2 / DR5, DRS, EGF L7, EGFR, EGFRvIII, endoglin, ENPP3, EpCAM, EphA 2. Episialin, FAP, FGFR1, FGFR2, FGFR3, FGFR4, Fib Ronectin extra domain B, FLT-3, flt4, folate receptor 1, guanylylsilane Clase C (GCC), GD2, GD3, glypican-3, glypican, GM3, GPN MB, GPR49, GRP78, Her2 / Neu, HER3 / ERBB3, HLA-D R, ICAM-1, IGF-1R, IGFR, IL-3Ra, integrin α5β1, Integrin α6β4, integrin αV, integrin αVβ3, Lewis Y, Lewis y / b antigen, LFL2, LIV-1, Ly6E, MCP-1, mesothelin, MMP-9, MU C1, MUC18, MUC5A, MUC5AC, myostatin, NaPi2b, Neuro Pyrin1, NGcGM3, NRP1, P-cadherin, PCLA, PD-1, PDGFR a, PD-L1, PD-L2, phosphatidylserine, PIVKA-II, PLVAP, PRLR, Progastrin, PSCA, PSMA, RANKL, RG1, Siglec- 15, SLAMF6, SLAMF7, SLC44A4, STEAP-1, TACSTD- 2. Tenascin-C, TPBG, TRAIL-R1 / DR4, TROP-2, TWEAKR , TYRP1, VANGL2, VEGF, VEGF-C, VEGFR-2 or VEG The present invention includes an antibody or an antigen-binding fragment thereof that binds to F-R2.

[0072] In some embodiments, the first and / or second targeting moiety is an anti-α4 integrin. Phosphoantibody, anti-CD137 antibody, anti-CCR4 antibody, anti-CD123 antibody, anti-CD133 antibody, Anti-CD138 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody , anti-CD38 antibody, anti-CD40 antibody, anti-CD49d antibody, anti-CD52 antibody, anti-CD70 antibody body, anti-CD74 antibody, anti-CD79b antibody, anti-CD80 antibody, anti-CEA antibody, anti-cMet antibody body, anti-Cripto antibody, anti-CTLA-4 antibody, anti-DLL3 antibody, anti-TRAIL-2 / D R5 antibody, anti-E-cadherin antibody, anti-endoglin antibody, anti-EpCAM antibody, anti-epithelial growth factor antibody Factor receptor antibody, anti-FGFR3 antibody, anti-fibronectin extra domain B antibody, anti Folate receptor 1 antibody, anti-glypican 3 antibody, anti-gp95 / 97 antibody, anti-Her2 antibody, anti-I GF-1R antibody, anti-IL-13R antibody, anti-IL-4 antibody, anti-IL-6 antibody, anti-MMP-9 Antibody, anti-MUC1 antibody, anti-mucin core protein antibody, anti-NGcGM3 antibody, anti-P-cad Herin antibody, anti-PD-L1 antibody, anti-p-glycoprotein antibody, anti-PSCA antibody, anti-PSMA Antibody, anti-SLAMF7 antibody, anti-TRAIL-R1 / DR4 antibody, anti-transferrin antibody an antibody, or an antigen-binding fragment thereof, which is an anti-TROP-2 antibody or an anti-VEGF antibody, include.

[0073] In some embodiments, the first and / or second targeting moiety is alemtuzumab. , Andecaliximab, Atezolizumab, Avelumab, BCD-100, Bevacizumab, BGB-A317, blinatumomab, brentuximab, BU59, camrelizumab, Rotuximab, Catumaxomab, Cemiplimab, Cetuximab, Daratumumab, Depatuki Cisumab, dinutuximab, DS-8201, durvalumab, edrecolomab, elotuzumab Zumab, G544, Gemtuzumab, Glembatumumab, GP1.4, hp67.6, IB I308, Ibritumomab, Inotuzumab, Ipilimumab, Isatuximab, L19IL 2. L19TNF, margetuximab, mirvetuximab, mogamulizumab, moxetum Mab, natalizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, Lartumab, oportuzumab, panitumumab, PDR001, pembrolizumab, pertz izumab, polatuzumab, racotumomab, ramucirumab, rituximab, rovalpituzumab , sacituzumab, SM3, TAK-164, tositumomab, trastuzumab, tremelim Mab, ublituximab, urelumab, utomilumab, XMAB-5574, or sol Includes betuximab.

[0074] In some embodiments, the first and / or second targeting moiety is an IL-2, IL -4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, I L-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, the first and / or second targeting moiety is selected from the group consisting of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL-1 3. Contains the full-length sequence of stem cell factor, insulin-like growth factor (IGF) or CD40. In some embodiments, the first and / or second targeting moiety is an IL-2, IL- 4. IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL -13, stem cell factor, truncated form of insulin-like growth factor (IGF) or CD40, related This includes any equivalents, variants or derivatives.

[0075] In some embodiments, the first and / or second targeting moiety is an IL-2 receptor. , IL-4, IL-6, melanocyte-stimulating hormone receptor (MSH receptor), transfusion Folate receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1 ), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR or binds to CD40L.

[0076] In some embodiments, the second targeting moiety is expressed by tumor microenvironment cells. In some embodiments, the tumor microenvironment cells are fibroblasts or macrophages. In some embodiments, the antigen expressed by the fibroblast is a fibroblast phage. In some embodiments, the IL-11A is a cell activation protein expressed by macrophages. The antigens used are MAC-1 / CD11b or sideroflexin 3.

[0077] The present application also provides a method for administering a first targeting moiety and / or a second targeting moiety to a patient. A method for treating a cancer expressing a tumor antigen that binds to the compound comprising administering to a patient a composition comprising: In some embodiments, administering the composition comprises administering a Prior to administering the composition, the cancer is evaluated for the presence of infiltrating immune cells. Prior to administering the antibody, the cancer is evaluated for the presence of a tumor antigen. The one targeting moiety and the second targeting moiety bind to the same antigen. In the above, the first targeting moiety and the second targeting moiety bind to different antigens.

[0078] In some embodiments, the tumor antigen that binds to the first and / or second targeting moiety Cancers in which it is expressed include breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, and melanoma. Cancer, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, stomach cancer, pancreatic cancer, colon cancer, liver cancer Pancreatic cancer, leukemia, myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, acute Myelodysplastic disorders, chronic lymphoblastic leukemia, lymphoproliferative disorders, myelodysplastic disorders, It is one of the following: myeloproliferative disorders or pre-malignant disorders.

[0079] In some embodiments, both the first and / or second targeting moieties in the patient The method of targeting immune cells to a cancer expressing a tumor antigen that binds to the composition comprises administering the composition to a patient. In some embodiments, one tumor antigen in the patient is a first A tumor antigen binds to a targeting moiety and a second tumor antigen binds to a targeting moiety. The method of targeting immune cells to a cancer expressing two tumor antigens includes administering a composition administering to a patient.

[0080] In some embodiments, the method of delivering a cytokine to immune cells of a patient comprises administering a composition administering to the patient, wherein the first and / or second supplemental functional domains of the composition comprise: IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ , or members of the TNF superfamily.

[0081] Additional objects and advantages will be set forth in part in the description which follows and in part will be apparent from the description. The objects and advantages of the present invention will become apparent or may be learned by practice. This will be realized and attained by means of the elements and combinations specifically recited within the accompanying drawings.

[0082] Both the foregoing general description and the following detailed description are exemplary and explanatory only. It should be understood that this is not intended to limit the scope of the claims.

[0083] The accompanying drawings, which are incorporated in and constitute a part of this specification, show one or more The embodiment(s) illustrated herein are intended to illustrate, together with the description, the principles described herein. It helps to clarify. [Brief description of the drawings]

[0084] [Figure 1A] 1 shows the twin immune cell engager ("TWICE"). The two constructs are shown prior to administration to a patient. [Figure 1B]1 shows a twin immune cell engager ("TWICE"). TWICE is shown bound and engaged to two signaling molecules on an effector (immune) cell after directing both components of TWICE to the cancer cell. The hexagons and diamonds represent two different antigens on the cancer cell bound to the targeting moieties of the first and second components (two sets of four hatched ovals each). Each component contains an immune cell binding domain (black domain of each component) and a complementary binding domain (white domain of each component). Linkers between the targeting moieties and the immune cell binding domain and complementary binding domain are also shown. When the two components are in close proximity, domain swapping can occur such that the two immune cell binding domains (one from each component) and the two complementary binding domains (one from each component) can pair with each other. The triangles and circles represent different antigens on the effector cell that can be bound either by the paired immune cell binding domain (bound to the antigen represented by the triangle) or by the paired complementary binding domain (bound to the antigen represented by the circle). In the absence of domain swapping and pairing, the immune cell binding domain and complementary binding domain of the two components do not modulate the effector cell. [Diagram 2]TWICE is shown engaging two signaling molecules on an effector (immune) cell after targeting of the first component to a cancer cell and the second component to a non-cancer cell. The hexagon represents an antigen on a cancer cell bound to the targeting moiety of the first component (four hatched ovals). The square represents an antigen on a non-cancer cell in the tumor microenvironment (e.g., on a tumor-associated macrophage, or on a fibroblast) bound to the targeting moiety of the second component (four hatched ovals). Each component contains an immune cell binding domain (black domain of each component) and a complementary binding domain (white domain of each component). When two components are in close proximity, domain swapping can occur such that two immune cell binding domains (one from each component) and two complementary binding domains (one from each component) can pair with each other. The triangles and circles represent different antigens on the effector cell that can be bound by the paired immune cell binding domain (antigen represented by the triangle) and the paired complementary binding domain (antigen represented by the circle). In the absence of domain swapping and pairing, the immune cell binding domain and complementary binding domain of the two components do not modulate the effector cell. [Diagram 3]Figure 2 shows TWICE binding and engaging signaling molecules on two different effector (immune) cells after both components of TWICE are targeted to the cancer cell. Various targeting strategies can be employed. When TWICE has two components that bind to the cancer cell, it can adopt three different configurations: (a) the two components of TWICE can bind to the same antigen using two copies of the same targeting moiety (single antigen targeting), (b) TWICE can use a single antigen strategy using different targeting moieties that target different epitopes on the same antigen, or (c) the two components of TWICE can bind to two different antigens upon binding of both targeting moieties to the cancer cell (dual antigen targeting). This figure represents single antigen targeting using different targeting moieties that target different epitopes on the same antigen. Returning to the figure, the diamonds represent two molecules of the same antigen on the cancer cell bound to the targeting moieties of the first and second components (two sets of four hatched ovals). Each component contains an immune cell binding domain (black domain of each component) and a complementary binding domain (white domain of each component). When two components are in close proximity, domain swapping can occur such that two immune cell binding domains (one from each component) and two complementary binding domains (one from each component) can pair with each other. The triangles and circles represent antigens on two different effector cells that can be bound to either the paired immune cell binding domain (bound to the antigen represented by the triangle on effector cell #1) or the paired complementary binding domain (bound to the antigen represented by the circle on effector cell #2). In the absence of domain swapping and pairing, the immune cell binding domains and complementary binding domains of the two components do not regulate the effector cells. [Figure 4]TWICE is shown binding and engaging with signaling molecules on effector (immune) cells after directing both components of TWICE to cancer cells, as well as binding to signaling molecules on cancer cells. The diamonds represent two molecules of the same antigen on cancer cells bound to the targeting moieties of the first and second components (two sets of four hatched ovals). Each component contains an immune cell binding domain (black domain of each component) and a complementary binding domain (white domain of each component). When the two components are in close proximity, domain swapping can occur such that two immune cell binding domains (one from each component) and two complementary binding domains (one from each component) can pair with each other. The triangles represent antigens on effector cells that can be bound to the paired immune cell binding domains. The hatched rectangles represent antigens on cancer cells that can be bound to the paired complementary binding domains. In the absence of domain swapping and pairing, the immune cell binding domains and complementary binding domains do not modulate the effector cells or cancer cells. [Diagram 5]TWICE including functional domains is shown before administration to a patient. The targeting moieties of the first and second components (two sets of four hatched ovals each) are shown. Each component includes an immune cell binding domain (black curved domain of each component). Each component also includes an inactive binding domain (also known as an inactive binding partner, white curved domain of each component) bound to the immune cell binding domain. One component also includes a supplemental functional domain (black triangle). Linkers between the targeting moiety and the immune cell binding domain or the supplemental functional domain are also shown. In addition, linkers between the immune cell binding domain and the inactive binding partner of each component are shown. The linker connecting the inactive binding partner can be a cleavable linker that is cleaved in the tumor microenvironment. This cleavage releases the inactive binding partner. When the two components are in close proximity and the inactive binding partner is released, domain exchange can occur such that the two immune cell binding domains (one from each component) can pair with each other. In addition, the complementary functional domain can bind and engage a target cell based on the targeting moiety localizing it to the target cell (e.g., a cancer cell or an effector cell). The action of the complementary functional domain occurs without the need for pairing with another domain and is enhanced when the complementary functional domain is localized to the target cell by the targeting moiety. In the absence of domain exchange and pairing, the immune cell binding domains of the two components do not modulate the target cell. In some embodiments, both components may include complementary functional domains. In some embodiments, only one component includes an inactive binding partner. [Figure 6]TWICE including the dimerization domain is shown before administration to a patient. The targeting moieties of the first and second components (two sets of four hatched ovals each) are shown. Each component includes an immune cell binding domain (black domain in each component) and a complementary binding domain (white domain in each component). Linkers between the targeting moieties and the immune cell binding domain and the complementary binding domain are also shown. The dimerization domain is linked to the immune cell binding domain and the complementary binding domain of each component by a linker (such that each component has a dimerization domain A and a dimerization domain B that can associate). The association of a pair of dimerization domains (pairing of dimerization domain A with dimerization domain B) enhances the association of the VH and VL domains of each component. The dimerization domain linker can be cleavable such that the linker is cleaved in the tumor microenvironment and the dimerization domain is lost after cleavage. After the dimerization domain is removed, and when the two components are brought into close proximity, domain swapping can occur such that the two immune cell binding domains (one from each component) and the two complementary binding domains (one from each component) can pair with each other. In the absence of domain swapping and pairing, the immune cell binding domains and complementary binding domains of the two components do not modulate the target cell. In the absence of cleavage of the dimerization domain linker, the VH and VL domains of each component remain associated with each other without domain swapping. In some embodiments, only one component contains a pair of dimerization domains, and the other component does not contain a dimerization domain. [Figure 7A] Figure 1 shows expression and cleavage of TWICE molecules listed in Table 12 with various dimerization domain linkers and dimerization domains.Figure 1 shows TWICE with various dimerization domain linkers and dimerization domains. [Figure 7B] Expression and cleavage of TWICE molecules listed in Table 12 with various dimerization domain linkers and dimerization domains are shown. TWICE was analyzed by SES-PAGE. [Figure 7C] Expression and cleavage of TWICE molecules listed in Table 12 with various dimerization domain linkers and dimerization domains are shown. The cleavage products of TWICE were also analyzed. [Figure 8] AB show target binding by TWICE containing Fab or dsFv binding moieties at various concentrations (A) or by EC50 measurements (B). [Figure 9A] 1 shows a bridging ELISA of CD3 / CTLA4 TWICE with heterologous Fc dimerization domains. Assay design is shown. [Figure 9B] Figure 1 shows a cross-linking ELISA of CD3 / CTLA4 TWICE with heterologous Fc dimerization domains. [Figure 9C] Figure 1 shows a cross-linking ELISA of CD3 / CTLA4 TWICE with heterologous Fc dimerization domains. Figure 2 shows the results of CTLA-4 cross-linking. [Figure 10A] 10A-10B show the results of a CD3 bridging ELISA for CD3 / CTLA4 TWICE, which has a coiled-coil dimerization domain. [Figure 10B] Shown are the results of a CTLA4 bridging ELISA (FIG. 10B) for CD3 / CTLA4 TWICE, which has a coiled-coil dimerization domain. [Figure 11A] Results of CD3 bridging ELISA for CD28 / CD3 TWICE are shown. [Figure 11B] Results of the CD28 bridging ELISA (FIG. 11B) for CD28 / CD3 TWICE are shown. [Figure 12A] 1 shows the bridging ELISA of selralizumab (anti-CD28) and urelumab (anti-4-IBB) TWICE. Specifically, this shows the results of CD28 / 4-1BB (CD137) TWICE in the CD28 bridging ELISA. [Figure 12B] 1 shows a bridging ELISA using celalizumab (anti-CD28) and urelumab (anti-4-IBB) TWICE. Specifically, this shows the results of CD28 / 4-1BB (CD137) TWICE in a 4-1BB bridging ELISA. [Figure 13]Figure 1 shows PD-L1 cross-linking of Atezolizumab / Ipilimumab TWICE. Specifically, this shows the results of PD-L1 / CTLA-4TWICE with a PD-L1 cross-linking ELISA. [Figure 14A] Figure 1 shows T cell targeting by CD3 / CTLA4 TWICE with pre-cleavage on HCT-15 cells. Activation by pre-cleavage. [Figure 14B] 1 shows T cell targeting redirection by CD3 / CTLA4 TWICE with or without prior cleavage on HCT-15 cells. Experimental results with or without prior cleavage are shown. [Figure 15A] 1 shows the results of a cleavage assay demonstrating T cell targeting and killing of HCT15 cells by CD3 / CTLA-4 TWICE. [Figure 15B] Figure 1 shows T cell targeting and killing of HCT15 cells by CD3 / CTLA-4 TWICE. Interferon gamma release is shown. [Figure 15C] Figure 1 shows T cell targeting and killing of HCT15 cells by CD3 / CTLA-4 TWICE. Figure 2 shows the results of LDH release, a measure of specific lysis of HCT15 cells under different conditions. [Figure 15D] Figure 1 shows T cell targeting and killing of HCT15 cells by CD3 / CTLA-4 TWICE. Figure 2 shows the results of LDH release, a measure of specific lysis of HCT15 cells under different conditions. [Figure 16] Figure 1 shows T cell targeting by CD3 / CD28 TWICE against HCT-15. This figure shows HCT-15 cell killing by CD3 / CD28 TWICE as measured by LDH release. [Figure 17A] 1 shows homing of pre-activated / exhausted peripheral blood mononuclear cells (PMBCs) towards HCT15 cells as measured by flow cytometry. [Figure 17B] Homing of pre-activated / exhausted peripheral blood mononuclear cells (PMBCs) towards HCT15 cells as measured by interferon gamma release (FIG. 17B). [Figure 18A]NK cell homing towards HCT15 cells as measured by CD16a cross-linking ELISA. [Figure 18B] Shown is the homing of NK cells to HCT15 cells as measured by LDH release (FIG. 18B).

[0085] Sequence description Table 1 provides a list of certain sequences referenced herein.

[0086] [Table 1-1]

[0087] [Table 1-2]

[0088] [Table 1-3]

[0089] [Table 1-4]

[0090] [Table 1-5]

[0091] [Table 1-6]

[0092] [Table 1-7]

[0093] [Table 1-8]

[0094]

Table 1-9

[0095]

Table 1-10

[0096]

Table 1-11

[0097]

Table 1-12

[0098]

Table 1-13

[0099]

Table 1-14

[0100]

Table 1-15

[0101]

Table 1-16

[0102]

Table 1-17

[0103]

Table 1-18

[0104] [Table 1-19]

[0105] [Table 1-20] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] I. Gemini Immune Cell Engagers The Twin Immune Cell Engager ("TWICE") is a two-component complex consisting of two components. The term refers to a two-component complex in which the two domains pair and are capable of binding and engaging immune cells. Thus, a single TWICE contains two components, which are separated by two distinct It can exist as a single polypeptide or as a single polypeptide. The CE may be a single construct or it may be a two-component kit. Throughout this application and the claims, the use of the term TWICE specifically refers to Unless expressly stated otherwise, both embodiments are encompassed. We report that TWICE mediates various anticancer effects by activating various pathways. can be done.

[0107] As described in this application, TWICE comprises a targeting moiety, an immune cell binding domain, and The first and second components may each comprise a complementary binding domain. The targeting portion of the component directs TWICE to cancer cells or cells in the tumor microenvironment. When immune cell binding domains from both components pair with each other and / or When complementary binding domains from the components pair, these paired domains bind to the immune system. Functioning to regulate cellular activity or performing other (i.e., complementary) functions For example, paired complementary binding domains can block immune checkpoints. or may cause cell death of cancer cells.

[0108] The presence of the complementary binding domain in TWICE is distinct from the complex that does not contain the complementary domain. In comparison (i.e., compared to complexes that only bind to a single antigen on an immune cell), This is expected to improve their effectiveness in treatment.

[0109] As also described herein, the first component, and optionally the second component, may be an immunoglobulin. The polypeptide may contain a supplementary functional domain capable of binding to a cell. In some embodiments, the immune cell binding domain is masked. Complementary functional domains of E regulate immune function without requiring pairing with distinct domains It is possible.

[0110] The main advantage of the TWICE complexes of the invention described herein is that the first and second When both components are in close proximity (where they both target cancer), Therefore, only when present in the tumor microenvironment are immune cell-binding domains and several The point is that in some embodiments, complementary binding domains will pair. Domain pairing between different components of TWICE at the off-position may improve specificity, It may reduce the targeting effect. The binding domains are expressed in the cancer cells or their microenvironment after pairing of domains from both components. This is because it only works for

[0111] We will first describe some of the most popular types of TWICE, and then introduce the different types of TWICE. The compositions comprising TWICE are described subsequently in this application. TWICE can regulate many pathways involved in the response, and thus is an important alternative to the alternatives described herein. It is not limited to the obvious.

[0112] The TWICE complex of the present invention is a two-component complex that activates multiple functions in the tumor microenvironment. Thus, the complexes of the present invention provide a unique ability to integrate into the tumor microenvironment. The two-component complex has the ability to be localized and induce two distinct signals to benefit patients. Having a single approach to providing the same results provides significant advantages. In this embodiment, one of the signals (from the paired immune cell binding domains) It is only activated when the two-component complex assembles. In some embodiments, and their complementary binding domains (from the paired immune cell binding domains). Both signals are activated only when the two-component complex is assembled. This unique construct , providing advantages not available in prior art constructions.

[0113] 1. TWICE activates two signaling pathways in one type of immune cell In some embodiments, the immune cell binding domains when paired and the paired When mated, the complementary binding domains bind to the same type of immune cell.

[0114] As used herein, immune cells are sometimes referred to as "effector cells." The term "effector cell" refers to an immune cell that mediates an effect against cancer. .

[0115] In some embodiments, the immune cell binding domains when paired and the paired The complementary binding domains when mated bind to T cells.

[0116] In some embodiments, the immune cell binding domains when paired and the paired When combined, the complementary binding domains bind to the same antigen on immune cells. In an embodiment, the two signaling pathways are the same and the effect is amplified. In embodiments, the immune cell binding domains when paired and the The complementary binding domains when combined bind to different antigens on immune cells. The two signaling pathways are distinct, and two different signals are transmitted to immune cells. will be done.

[0117] In general, the process of T cell activation involves multiple signals. Major histocompatibility complexes presented by the T cell receptor (TCR) and antigen-presenting cells (APC) The costimulatory signal comes from binding to major histocompatibility complex (MHC) molecules. This may result from one of the interactions between the cells and the APC (Buchbinder EI, Desai See Am J Clin Oncol. 39(1):98-106(2016) T cell activation is mediated by several costimulatory receptors, e.g., CD28, CD13 7(4-1BB), OX40, CD27, GITR(TNFRSF18) and ICOS The interaction between T cells and APCs or cancer cells also enhances T cell activation. Several inhibitory / checkpoint molecules, e.g., PD-1, CTLA-4, , TIM-3, LAG-3 and KIR are involved (Torphy RJ et al. nt J Mol Sci.18:2642(2017)). Therefore, co-stimulation or co- Combining the effects of two antibodies against inhibitory receptors enhances antitumor T cell responses It is possible.

[0118] TWICE reacts (after pairing) to costimulatory or checkpoint molecules on T cells. The antibody may contain two functional antibodies that interact with each other, leading to robust T cell activation (Figure 2). Pairing of immune cell binding domains of the two components, and pairing of the white complementary binding domains of the two components. Robust T cell activation was observed when T cells were present in the tumor but not isolated from the cancer cells (Figure 1B). These findings may be useful for patients whose immune system is inactive due to immune-suppressive signals from other immune systems (Wu AA et al. OncoImmunology 4:7(2015)).

[0119] In some embodiments, the immune cell binding domains when paired and the paired When combined, the complementary binding domains mediate agonistic or antagonistic binding to T cells. Thus, it can engage immune cells.

[0120] "Agonistic" refers to the binding of a paired immune cell binding domain or a paired complementary binding domain. This means that the binding of the antigen to the immune system activates the signal transduction pathway. Efficient binding may be of interest if it leads to immune cell activation.

[0121] "Antagonistic" refers to the ability of paired immune cell binding domains or paired complementary binding domains to bind to one another. This means that the binding of the antigen to the antigen blocks or inhibits the signal transduction pathway. Antagonistic binding is of interest when normal binding of a ligand to a target leads to immune cell inactivation. Thus, the binding of an "antagonistic" set of paired domains of TWICE of the present invention is By blocking or inhibiting pathways that normally mediate immune cell unresponsiveness or inactivation It can activate immune cells.

[0122] The antibodies known to have antagonistic or agonistic effects on immune cells are used in the present invention. can be used to generate paired binding domains of TWICE. If the signaling pathway promotes T cell activation, then agonistic approaches could be used to increase T cell activation. If the signaling pathway is one that inhibits T cell activation, then antagonistic Thus, a variety of pathways could be used in this approach to increase T cell activation. It can be adopted.

[0123] An example of an agonist antibody is a CD3 epsilon antibody (e.g., SP34) that stimulates the TCR complex. In another example, an agonist antibody against GITR inhibits regulatory T cell function. and activate CD8+ T effector cells (Knee et al. l.,European Journal of Cancer 67:1e10(20 16) Antagonistic antibodies that lead to stimulation of immune cells include PD-1 antibodies (e.g. PD-1 suppresses immune cells. Thus, these antibodies block the interaction of PD-1 with its ligands, thereby This antagonizes inhibitory signals.

[0124] In some embodiments, the immune cell binding domains when paired and the paired The complementary binding domains when combined can generate two agonistic antibodies, resulting in an immunosuppressive effect. It can provide two positive stimuli to the immune system (e.g., anti-CD3 epsilon and anti-CD1 37 antibodies). Both CD3 and CD137 promote T cell activation.

[0125] In some embodiments, the immune cell binding domains when paired and the paired The complementary binding domains when combined form one agonist antibody and one antagonist antibody (e.g. For example, anti-CD3 epsilon and anti-CTLA4 antibodies can be produced. While stimulating the TCR complex as described above, CTLA4 provides an inhibitory signal. Antagonizing the inhibitory signal of CTLA4 promotes T cell activation.

[0126] In some embodiments, the immune cell binding domains when paired and the paired When combined, the complementary binding domains form two antagonistic domains that block two inhibitory signals. Antibodies (e.g., anti-CTLA4 antibodies and anti-PD-1 antibodies) can be produced. As previously reported, both of these signals inhibit T cell activation and their antagonism This will promote T cell activation.

[0127] Another exemplary TWICE promotes CD3 epsilon (CD3e) antibody and T cell activation The current evidence supports the use of CD3 bispecific antibodies in combination with another antibody that targets the These results suggest that the disease is further regulated by various checkpoint molecules (Kobol d See S et al. Front Oncol. 8:285(2018) In addition, the therapeutic effect of bispecific antibodies that activate T cells may be limited in some patients or situations. In this study, T cell anergy (absence of normal immune responses to specific antigens) was induced. This may be due to the presence of anti-CD3 ipsin which activates T cells. By combining a pro-inflammatory antibody with an antibody that enhances T cell activation and prevents unresponsiveness, Enhanced and / or more durable anti-tumor T cell responses may result.

[0128] 2. TWICE regulates two types of immune cells In some embodiments, the immune cell binding domains when paired and the paired When combined, the complementary binding domains bind to two different immune cells. Thus, TWICE can regulate two different types of immune cells. A variety of immune cells can be regulated by TWICE. Figure 2 shows the TWICE-mediated 2 Representative activation models of two immune cells (effector cell #1 and effector cell #2) Indicates the rule.

[0129] a) TWICE stimulates T cells and suppresses tumor-associated macrophages The binding of two different immune cells allows T cells to be expelled from the tumor and transformed into tumor-associated macrophages ( TAMs may be useful in the tumor microenvironment when they are present. Alternatively activated IL-1 receptors promote tumor growth by promoting angiogenesis, immunosuppression, and inflammation. The macrophage class that has been identified (Poh and Ernst, Front Onco l.12(8):49(2018)). In some embodiments, TWICE is complementary By blocking TAM activity when the binding domain and the immune cell binding domain are paired. It combines the effects of immunization with T cell activation.

[0130] In some embodiments, TWICE mediates T cell activation when paired. and a complementary binding domain that inhibits TAMs when paired. Includes

[0131] For example, targeting anti-CSF1R blocking antibodies to TAMs reduces TAM infiltration. and promotes CD8+ T cell proliferation (Ries CH et al. Cancer Cell.25(6):846-59(2014)). Activation of the CD40 pathway also promotes TAM CD40 agonist antibodies can counteract the immunosuppressive effects of classically activated myeloid leukemia. This can result in the reprogramming or recruitment of phage to tumors. These antibodies can be used to induce effective immune responses that stimulate T cell activation and CD40 agonist antibodies. Representative TWICE in combination with an anti-CSF1R blocking antibody is and after pairing of the complementary binding domains, the additional This may provide practical effectiveness.

[0132] b) TWICE stimulates T cells and NK cells TWICE activates both T cells and NK cells that kill cancer cells. Coactivation of T cells and NK cells can be used to activate either cell alone. This could lead to a more robust immune response against cancer compared to conventional immunotherapy.

[0133] In some embodiments, TWICE mediates T cell activation when paired. Immune cell binding domains that mediate NK cell activation when paired Complementary binding domains include, for example, anti-CD16A antibodies. For example, TWICE440 and T In TWICE, which includes WICE441, anti-CD16A antibody is combined with anti-CD3 antibody OKT3. (See SEQ ID NOs:212-215 and Figures 18A-18B).

[0134] 3. TWICE stimulates immune cells and mediates effects against cancer cells TWICE binds to immune cells via paired immune cell-binding domains, and The complementary binding domains of the mAb and mAb can also be paired with each other to bind to cancer cells. In addition to mediating anti-cancer immune cell activation, these strategies aim to induce biological mechanisms in targeted cancer cells. The biological functions induced in cancer cells are e.g. For example, it may be the induction of cell death via a death receptor, or the inhibition of immunosuppressive signals.

[0135] Paired immune cell binding domains capable of activating immune cells have been described. and the TWICE component may also contain a complementary binding domain that binds to the cancer.

[0136] a) TWICE stimulates immune cells and prevents immune cell inactivation by cancer The TWICE of the present invention activates immune cells and inhibits immune cells from cancer cells. In some embodiments, the complementary The specific binding domain inhibits immune cell activation from cancer cells when paired with each other. The signal may be blocked.

[0137] For example, some cancers express PD-L1, which binds to PD-1 on T cells. or B7.1 and can lead to T cell inactivation. In some embodiments, When paired, the complementary binding domains bind to PD-L1 on cancer cells. In some embodiments, binding to PD-L1 can be achieved by binding to PD-1 on T cells. Blocking the interaction between PD-1 and PD-L1 improves antitumor responses. PD-L1 antibodies (e.g., atezolizumab, durvalumab, and Avelumab) is used for the treatment of several malignant diseases, including non-small cell lung cancer and melanoma. has been approved.

[0138] In another example, some cancers express the tumor necrosis factor receptor (TNFR) family. These molecules may downregulate the anti-tumor immune response. Receptor activating nuclear factor kappa B (RANK), which is its ligand on regulatory T cells. It binds to the oncolytic enzyme RANKL, creating an immunosuppressive environment (Wu et al. In some embodiments, When paired, the complementary binding domains bind to RANK on cancer cells. In some embodiments, interaction with RANKL on T cells by binding to RANK. In preclinical mouse models of cancer, antagonistic antibodies against RANKL Blockade of RANK signaling enhances the antitumor effect of anti-CTLA4 antibodies, which in turn enhances the T cell (Ahern et al. Clin Cancer Res. 23( 19):5789-5801(2017)).

[0139] b) TWICE stimulates immune cells and induces cell death in cancer cells TWICE of the present invention activates immune cells and induces cell death in cancer cells In some embodiments, the complementary binding domains may be used for both: When paired together, they bind to receptors expressed by cancer cells. It can induce the death of cancer cells.

[0140] Examples of receptors that can be stimulated to induce cell death include the TNFR family of receptors. Members such as Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, D R5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER or CAR1 are listed. TNFR family agonist antibodies in clinical development that induce cell death in cancer cells The drug is mapatumumab (NCT01258608), which has the effect of directly killing cancer cells. In combination, TWICE, which activates anti-cancer immune responses, may have additive efficacy.

[0141] 4. TWICE containing supplementary functional domains The first and second components of TWICE may also comprise complementary functional domains. The "complementary functional domains" used act as a complement to other domains when directed to the appropriate immune cells. TWICE refers to a molecule that regulates immune function without the need for pairing with a ligand. Supplementary functional domains can be targeted to immune cells, which lack activity without targeting. or to stimulate the action of complementary functional domains that may be too toxic for systemic delivery. Exemplary supplemental functional domains can be activated by appropriate immune cells to have activity. This embodiment is shown in FIG. Attenuated cytokines are IL-2 pathway agonists with a preference for CD122 As shown by the data on Bempegaldesleukin (NKTR-214), Target cells can be preferentially activated.

[0142] Thus, TWICE mediates the action and complementary functionality of paired immune cell binding domains. The action of these domains can mediate additional anti-cancer immune responses. Effector cells are activated only at the effector cell / cancer cell engaging synapse. The ministry will deliver a "medical package" to

[0143] B. A single polypeptide chain or two components TWICE may comprise a single polypeptide chain, or two separate components.

[0144] In some embodiments, the first component is not covalently bonded to the second component. In some embodiments, the first moiety is covalently bonded to the second moiety.

[0145] In some embodiments, TWICE consists of two separate components. The ICE may consist of first and second components which are separate polypeptides.

[0146] In some embodiments, TWICE consists of a single polypeptide chain. In this embodiment, the first and second components are contained within a single amino acid sequence.

[0147] When TWICE consists of a single polypeptide chain, the first and second components are connected by a linker. In some embodiments, the linker may be a covalent linker between the first and second components. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the cleavable linker between the first and second components is a protease cleavable linker. Contains the cleavage site.

[0148] In some embodiments, a linker covalently attaches the first component and the second component. The cleavage sites contained within the sequences are protease cleavage sites. Several exemplary protease cleavage sites that can be used are listed below, with TWICE being one of them. The protease cleavage site is not limited to the above protease cleavage site, and other protease cleavage sites may be employed.

[0149] In some embodiments, a linker covalently attaches the first component and the second component. The cleavage site contained within the cleavage site is a tumor-associated protease cleavage site. In some embodiments, the proteinase is associated with a tumor. ase is more highly expressed in tumors than in other regions of the body. Examples of tumor-associated proteases are shown in the figure. Any protease expressed in the tumor may be used to select the cleavage site. In some embodiments, the protease inhibits the proliferation of non-cancer cells in the tumor microenvironment, such as tumor-associated myeloid cells. It is expressed by phages or fibroblasts.

[0150] In some embodiments, a linker covalently attaches the first component and the second component. The cleavage sites contained within the cleavage site are cleavage sites for proteases found in blood. Exemplary proteases found in blood include thrombin, neutrophil elastase, and and furin.

[0151] C. Targeting part The targeting moiety of the first component acts by delivering the drug to the local environment of the cancer cells. In some embodiments, the first targeting moiety is , which targets cancer cells by specifically binding to them.

[0152] In some embodiments, both the first and second targeting moieties are caused by cancer. It binds to expressed tumor antigens.

[0153] In some embodiments, the first targeting moiety targets a tumor antigen expressed by the cancer. and the second targeting moiety binds to an antigen expressed by tumor microenvironment cells.

[0154] In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. By antigen-binding fragment we mean an antigen-binding fragment that retains its binding activity for the target. Any antibody fragment having a light chain, such as an scFv or other functional fragment, e.g., a fragment not having a light chain. Immunoglobulins, VHH, VNAR, Fab, Fab', F(ab')2, Fv, antibodies Fragment, diabody, scAB, single domain heavy chain antibody, single domain light chain antibody, Fd, CDR regions, or any portion or peptide of an antibody capable of binding to an antigen or epitope. VHH and VNAR are alternatives to classical antibodies and Although are produced in different species (camel and shark, respectively), the inventors These are also included in the antigen-binding fragment of an antibody. In some embodiments, the Fv domain is a disulfide. When the present application refers to an antibody, it is specifically Unless "full-length antibody" is specified herein, the term essentially includes the meaning of an antigen-binding fragment thereof.

[0155] In some embodiments, the targeting moiety is not an antibody but another type of targeting moiety. A variety of targeting moieties are known that can target cancer, including: DNA aptamer, RNA aptamer, albumin, lipocalin, fibronectin, Nkirin, Finomer, Obody, DARPin, knotins, Bimers, atrimers, anticalins, affilins, affibodies, bicyclic peptides, cy s-knot, FN3 (adnectins, centryrins, pronec These and other non-antibody domains include cytochrome P450 (TN4), cytochrome P450 (TN5), and Kunitz-type domains. The scaffold structures can be used to target cancer cells. Smaller non-antibody scaffolds can be targeted to the bloodstream. They are rapidly cleared from the blood and have a shorter half-life than monoclonal antibodies. It exhibits faster tissue penetration due to faster extravasation from capillaries through the vascular endothelium and basement membrane. azquez-Lombardi et al.,Drug Discovery To See Day 20(1):1271-1283 (2015). Several non-antigen scaffolds that can be used to treat cancer are already in clinical development, and other candidates are in the preclinical stage. See Table 1 in Ez-Lombardi.

[0156] 1. Targeting moieties capable of targeting cancer In some embodiments, only the first targeting moiety targets an antigen expressed by the cancer. In some embodiments, the first and second targeting moieties bind to each other and the second targeting moiety does not bind to each other. Both of the second targeting moieties bind to antigens expressed by the cancer.

[0157] In some embodiments, the first and second targeting moieties are the same and bind to the same antigen. In some embodiments, the first and second targeting moieties are coupled to a cancer cell. In some embodiments, the first and second targeting moieties bind to the same expressed antigen. bind to different epitopes on the same antigen expressed by cancer. In embodiments, the first and second targeting moieties bind to different antigens expressed by the cancer. Combine.

[0158] Certain specific tumor antigens that may be used (examples of cancer cell types are in parentheses) include Herceptin, 2 / Neu (epithelial malignancies), CD22 (B-cell malignancies), EpCAM (CD326) (epithelial malignant tumors), EGFR (epithelial malignant tumors), PSMA (prostate carcinoma), CD30 (B cell malignancies), CD20 (B cell malignancies), CD33 (myeloid malignancies), CD80 ( B-cell malignancies), CD86 (B-cell malignancies), CD2 (T-cell or NK-cell lymphomas) CA125 (multiple cancers, including ovarian carcinoma), carbonic anhydrase IX (including renal cell carcinoma) CD70 (B-cell malignancies), CD74 (B-cell malignancies), CD5 6 (T-cell or NK-cell lymphoma), CD40 (B-cell malignancies), CD19 (B-cell malignant tumors), c-met / HGFR (digestive and hepatic malignant tumors, TRAIL-R1 / DR 4 (multiple malignant tumors including ovarian and colorectal carcinoma), DRS (multiple malignant tumors including ovarian and colorectal carcinoma) PD-1 (multiple malignancies, including B-cell malignancies), PD-L1 (multiple malignancies, including epithelial adenocarcinoma) tumors), IGF-1R (most malignant tumors, including epithelial adenocarcinoma), VEGF-R2 (epithelial adenocarcinoma) The vasculature associated with the majority of malignant tumors, including prostate cancer, is involved in the expression of prostate cell antigen (PSCA) ( cancer), MUC1 (epithelial malignancies), CanAg (tumors, e.g., colon and pancreatic carcinomas), Sothelin (mesothelioma and many tumors, including ovarian and pancreatic adenocarcinoma), P-cadherin (breast epithelial malignancies, including adenocarcinomas), myostatin (GDF8) (sarcomas and ovarian and pancreatic adenocarcinomas) Cripto (TDGF1) (in colon cancer, breast cancer, lung cancer, ovarian cancer, and many other cancers) and pancreatic cancer), ACVRL1 / ALK1 (leukemia and lymphoma MUC5AC (epithelial malignant tumors including breast cancer), CEACAM (breast epithelial malignancies, including adenocarcinomas), CD137 (B-cell or T-cell malignancies), CXCR4 (B-cell or T-cell malignancies), Neutropilin 1 (epithelial malignancies, including lung cancer), Glypican (multiple cancers, including liver, brain, and breast cancer), HER3 / ERBB3 ( epithelial malignant tumor), PDGFRa (epithelial malignant tumor), EphA2 (neuroblastoma, melanoma, breast CD38 (myeloma), CD138 (myeloma) , α4 integrin (AML, myeloma, CLL, and most lymphomas), CCR4 antibodies ( lymphoma), CD52 (leukemia), CD79b (lymphoma), CTLA-4 (non-small cell lung cancer) (multiple cancers, including urothelial carcinoma, head and neck cancer, urothelial carcinoma, and hepatocellular carcinoma), DLL3 (lung cancer), Indoglin (multiple cancers, including soft tissue sarcoma, angiosarcoma, and renal cell carcinoma), fibrone Cutin extra domain B (melanoma), folate receptor 1 (epithelial ovarian cancer, peritoneal carcinoma and fallopian tube cancer), MMP-9 (gastric cancer or gastroesophageal junction adenocarcinoma), NGcGM3 (lung cancer), SLAMF7 (multiple myeloma), TROP-2 (breast cancer), or VEGF (colon cancer) is one example.

[0159] In some embodiments, the targeting moiety is an alpha4 integrin, A33, ACVRL 1 / ALK1, ADAM17, ALK, APRIL, BCMA, C242, CA125, Cadherin-19, CAIX, CanAg, carbonic anhydrase IX, CCN1, CCR4, C D123, CD133, CD137(4-1BB), CD138 / syndecan-1, CD1 9, CD2, CD20, CD22, CD30, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD48, CD5, CD52, CD56, CD59, C D70, CD70b, CD71, CD74, CD79b, CD80, CD86, CD98 , CEA, CEACAM, CEACAM1, CK8, c-Kit, CLDN1, CLDN 18, CLDN18.2, CLDN6, c-met / HGFR, c-RET, Cript o, CTLA-4, CXCR4, DKK-1, DLL3, DLL4, TRAIL-R2 / DR5, DRS, EGFL7, EGFR, EGFRvIII, endoglin, ENPP3 , EpCAM, EphA2, episialin, FAP, FGFR1, FGFR2, FGFR 3. FGFR4, fibronectin extra domain B, FLT-3, flt4, folate Receptor 1, GCC, GD2, GD3, Glypican-3, Glypican, GM3, GPNMB , GPR49, GRP78, Her2 / Neu, HER3 / ERBB3, HLA-DR, ICAM-1, IGF-1R, IGFR, IL-3Ra, integrin α5β1, integrin integrin α6β4, integrin αV, integrin αVβ3, Lewis Y, Lewis y / b anti Hara, LFL2, LIV-1, Ly6E, MCP-1, mesothelin, MMP-9, MUC1 , MUC18, MUC5A, MUC5AC, myostatin, NaPi2b, neuropilin 1, NGcGM3, NRP1, P-cadherin, PCLA, PD-1, PDGFRa, PD-L1, PD-L2, phosphatidylserine, PIVKA-II, PLVAP, PR LR, progastrin, PSCA, PSMA, RANKL, RG1, Siglec-15 , SLAMF6, SLAMF7, SLC44A4, STEAP-1, TACSTD-2, Tenascin-C, TPBG, TRAIL-R1 / DR4, TROP-2, TWEAKR, T YRP1, VANGL2, VEGF, VEGF-C, VEGFR-2, or VEGF- Binds to R2.

[0160] Table 2 (shown in Section IV below) lists cancer types, possible targets for the targeting moieties, and their Non-limiting examples of proteases expressed by these cancer types are shown below. The TWIs may be referred to as tumor-associated proteases. To prepare a CE, a cancer may be identified and a target for a (desired) targeting moiety may be provided. For a cancer type or a desired tumor microenvironment cell (such as TAM or TAF), One or two proteases are selected. All TWICEs require protease cleavage. Although not required, protease cleavage may be performed as a single molecule with a cleavage site. This is beneficial in TWICEs that contain either an inactive binding partner or an inactive TWICE. obtain.

[0161] Table 3 (shown in Section IV below) describes cancers that can be targeted by different targeting moieties. This additional information demonstrates that some targeting moieties can target multiple different cancer types. In TWICE, the first component and the second component are Both components may include a targeting moiety capable of targeting cancer. and the targeting moieties of the second component may bind to the same antigen or different antigens.

[0162] Antibodies that bind to tumor antigens and have specificity for tumor cells are well known in the art. It is being done.

[0163] The FDA maintains a list of approved antibody drugs for treating cancer, many of which are , which bind to cancer antigens, may be employed in the context of the present invention. For more information, see The Orange Book Online or Drugs@FDA. FDA also has a database at clinicaltrials.gov that can be searched by disease name. The database also lists ongoing clinical trials. Table 4 (see Section IV below) Table 1 shows a representative list of approved antibodies with specificity for tumor cells. 5 (described in Section IV below) is representative of antibodies under development with specificity for tumor cells. A detailed list is provided below.

[0164] Table 6 (shown in Section IV below) shows the tumor antigen binding and targeting moieties of TWICE. This document compiles selected publications related to exemplary antibodies that can be used as antibodies. These publications demonstrate that targeting moieties capable of binding to tumor antigens are well within the skill of the art. This indicates that the subject matter disclosed herein was in the possession of the inventors at the time of filing of this application.

[0165] Other antibodies well known in the art can be used to target a given cancer. The antibody and its respective antigen may be used as a part. antibody), TA99 (anti-gp75), 3F8 (anti-GD2), 8H9 (anti-B7-H3), Ava Govomab (anti-CA-125 (mimetic)), adecatumumab (anti-EpCAM), aftuzumab (anti-CD20), aracizumab pegol (anti-VEGFR2), pentetic acid altumoma amatuximab (anti-CEA), amatuximab (anti-mesothelin), AME-133 (anti-CD20), Natumomab Mafenatox (anti-TAG-72), Apolizumab (anti-HLA-DR), Lucitumomab (anti-CEA), bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab (anti-BAFF), besilesomab (anti-CEA-related antigen), Cizumab (anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), Natumomab (anti-CD19), BMS-663513 (anti-CD137), brentuximab Vedotin (anti-CD30 (TNFRSF8)), Cantuzumab Mertansine (anti-mucin CanAg), cantuzumab ravtansine (anti-MUC1), capromab pendetide ( anti-prostate cancer cells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM,C D3), ​​cBR96-doxorubicin immune complex (anti-Lewis Y antigen), CC49 (anti-TA G-72), cedelizumab (anti-CD4), Ch.14.18 (anti-GD2), ch-TNT (anti-DNA-associated antigen), sitatuzumab bogatox (anti-EpCAM), cixutumumab ( anti-IGF-1 receptor), clivatuzumab tetraxetan (anti-MUC1), conatumumab (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab (anti-CD4 0), daclizumab (anti-CD25), dalotuzumab (anti-insulin-like growth factor I receptor) , daratumumab (anti-CD38 (cyclic ADP-ribose hydrolase)), demcizumab (anti DLL4), detumomab (anti-B lymphoma cells), dorozitumab (anti-DR5), durigozumab umab (anti-HER3), dusigitumab (anti-ILGF2), ecloneximab (anti-GD3 gas nglioside), edrecolomab (anti-EpCAM), elotuzumab (anti-SLAMF7), Elcilimomab (anti-IL-6), enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), encituximab (anti-5AC), epitumomab situxetan (anti-epi sialin), epratuzumab (anti-CD22), ertumaxomab (anti-HER2 / neu, CD3), etaracituzumab (anti-integrin αvβ3), faralimomab (anti-inter folate receptor), farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20) , Ficlatuzumab (anti-HGF), Fizitumumab (anti-IGF-1 receptor), Franbot Mab (anti-TYRP1 (glycoprotein 75)), fresolimumab (anti-TGFβ), Mab (anti-EGFR), Galiximab (anti-CD80), Ganitumab (anti-IGF-I), Gem Tuzumab ozogamicin (anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CA -IX), glembatumumab vedotin (anti-GPNMB), guselkumab (anti-IL13 ), ibalizumab (anti-CD4), ibiritumomab tiuxetan (anti-CD20), ikru Kumab (anti-VEGFR-1), igobomab (anti-CA-125), zolbetuximab (IM AB362, anti-CLDN18.2), IMC-CS4 (anti-CSF1R), IMC-TR1 (TGFβRII), imgatuzumab (anti-EGFR), inlacumab (anti-selectin P ), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin ( Anti-CD22), Intetumumab (anti-CD51), Ipilimumab (anti-CD152), Iratu Mumab (anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-076 1 (anti-CD194), LY2875358 (anti-MET), labetuzumab (anti-CEA), Embrolizumab (anti-PDCD1), lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirilumab (anti-KIR2D), lorvotuzumab mertansine (anti-C D56), lucatumumab (anti-CD40), rumiliximab (anti-CD23 (IgE receptor) ), mapatumumab (anti-TRAIL-R1), margetuximab (anti-ch4D5), matuz mab (anti-EGFR), mavrilimumab (anti-GMCSF receptor a chain), milatuzumab (anti-C D74), Minretumomab (anti-TAG-72), Mitsumomab (anti-GD3 ganglioside) , mogamulizumab (anti-CCR4), moxetumomab passudotox (anti-CD22), naclovir romab tafenatox (anti-C242 antigen), naptumomab estafenatox (anti-5 T4), narutomab (anti-RON), necitumumab (anti-EGFR), nesvacumab (anti-A 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4), nofe Tumor merpentan, ocrelizumab (anti-CD20), ocaratuzumab (anti-CD20 ), olaratumab (anti-PDGF-Rα), onartuzumab (anti-c-MET), ontuximab tumourab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab ( anti-CA-125), otlertuzumab (anti-CD37), vancomab (anti-tumor specific glycoprotein silylating MUC1), palsatuzumab (anti-EGFL7), pascolizumab (anti-IL-4), Patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab (anti-HER2 / neu), pidilizumab (anti-PD-1), pinatuzumab vedotin (anti-CD22), Ntumomab (anti-adenocarcinoma antigen), polatuzumab vedotin (anti-CD79B), pritumumab ( Anti-vimentin), PRO131921 (anti-CD20), Kirizumab (anti-IGHE), Laco Tumomab (anti-N-glycolylneuraminic acid), radretumab (anti-fibronectin exon tradomain B), ramucirumab (anti-VEGFR2), rilotumumab (anti-HGF), donkey Tumor tumour (anti-IGF-1 receptor), lorezumab (anti-RHD), and roberizumab (anti-CD11 and anti-CD18), samaryzumab (anti-CD200), satumomab pendetide (anti-TAG -72), seribantumab (anti-ERBB3), SGN-CD19A (anti-CD19), SG N-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab (anti-IL -6), solitomab (anti-EpCAM), sonituzumab (anti-episialin), tabalumab ( Anti-BAFF), tacatuzumab tetraxetan (anti-alphafetoprotein), tapri Tumomab paptox (anti-CD19), terimomab aritox, tenatumomab (anti-tenet Isin C), teneliximab (anti-CD40), teprotumumab (anti-CD221), TGN 1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab (anti-TRAI L-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20), tobetuma (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4), Melimumab (anti-CTLA-4), TRU-016 (anti-CD37), Tucotuzumab Selmo Leukin (anti-EpCAM), ublituximab (anti-CD20), urelumab (anti-4-1B B), vanticutumab (anti-Frizzled receptor), bapaliximab (anti-AOC3(V AP-1), batelizumab (anti-ITGA2), veltuzumab (anti-CD20), besenc Mab (anti-NRP1), visilizumab (anti-CD3), volociximab (anti-integrin α5 β1), borsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigen CTA A16.88), Zalutumumab (anti-EGFR), Zanolimumab (anti-CD4), Zatuximab (anti-HER1), diralimumab (anti-CD147 (basigin)), RG7636 (anti-E TBR), RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL 3280A (anti-PD-L1), RG7450 (anti-STEAP1), TAK-164 (anti-G CC), and GDC-0199 (anti-Bcl-2).

[0166] Antibodies that bind to these antigens have also been identified in TWICE, especially for the cancer types noted. May be used: aminopeptidase N (CD13), annexin A1, B7-H3 (CD 276, various cancers), CA125 (ovarian cancer), CA15-3 (carcinoma), CA19-9 ( L6 (carcinoma), Lewis Y (carcinoma), Lewis X (carcinoma), alpha-fetoprotein Plasma (carcinoma), CA242 (colon cancer), placental alkaline phosphatase (carcinoma), prostate specific Antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinoma), CD 2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma) , lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B cell malignant tumors) ), CD20 (non-Hodgkin's lymphoma, B-cell neoplasms, autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin lymphoma) lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD4 0 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma ), CD52 (leukemia), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma, and liquid tumors, multiple myeloma), CD66e (carcinoma), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (carcinoma), CD123 (leukemia), mucin (carcinoma), CD221 (solid tumors), CD22 (breast cancer, oocyte follicular cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 ( Solid tumors), CEACAM3 (colon cancer, gastric cancer), CEACAM5 (CEA, CD66e) (breast, colon and lung cancer), DLL4 (A-like 4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD184, hematologic oncology, solid tumors), endoglin (C D105, solid tumor), EPCAM (epithelial cell adhesion molecule, bladder cancer, brain tumor, cervical cancer, colon cancer) cancer, NHL prostate cancer and ovarian cancer), ERBB2 (lung cancer, breast cancer, prostate cancer), FCGR1 ( Autoimmune diseases), FOLR (Folate receptor, ovarian cancer), FGFR (carcinoma), GD2 ganglionic Osido (carcinoma), G-28 (cell surface antigen glycolipid, melanoma), GD3 idiotype (carcinoma), heat shock protein (carcinoma), HER1 (lung cancer, stomach cancer), HER2 (breast cancer, Lung and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia disease), human chorionic gonadotropin (carcinoma), IGF1R (solid tumors, blood cancer), IL-2 Receptor (T-cell leukemia and lymphoma), IL-6R (multiple myeloma, RA, Castleman disease, IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4 in various cancers , α11β3, α5β5, αvβ5), MAGE-1 (carcinoma), MAGE-2 (carcinoma), MAGE-3 (carcinoma), MAGE4 (carcinoma), anti-transferrin receptor (carcinoma), p9 7 (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, non-Hodgkin B-cell lymphoma, leukemia), MUC1 (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and gastrointestinal cancer) Organ cancer), MUC16 (CA125) (ovarian cancer), CEA (colon cancer), gp100 (melanoma melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domain insubfamily A, small cell lung cancer, NHL), nucleolin, Neu oncogene product ( Nectin-4 (carcinoma), anti-(N-glycolylneuraminic acid, breast paratope of PLAP-like testicular alkaline phosphatase (ovarian cancer, seminal prostate cancer), PSMA (prostate tumor), PSA (prostate), ROB04, TAG72 (tumor-associated glycoprotein 72, AML, gastric cancer, colon cancer, ovarian cancer), T cell transmembrane protein (cancer ), Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptor 10B) Parr family member 10B, carcinoma), TNFRSF13B (tumor necrosis factor receptor supramolecular Parr family member 13B, multiple myeloma, NHL, other cancers, RA and SLE), T PBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis Inducible ligand receptor 1, lymphoma, NHL, colon cancer, lung cancer), VCAM-1 (CD10 6, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers) Several other tumor-associated antigen targets have been reviewed (Gerber, et al. al,mAbs 2009 1:247-253, Novellino et al, Cancer Immunol Immunother.2005 54:187-20 7, Franke,et al,Cancer Biother Radiopharm .2000,15:459-76,Guo,et al.,Adv Cancer Re s.2013, 119:421-475,Parmiani et al.J Immu Nol. 2007 178:1975-9). Examples of these antigens include the cluster of differentiation antigens ( CD4, CDS5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11 b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49 c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD 62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, C D109, CD117, CD120, CD127, CD133, CD134, CD135 , CD138, CD141, CD142, CD143, CD144, CD147, CD1 51, CD152, CD154, CD156, CD158, CD163, CD166, C D168, CD184, CDw186, CD195, CD202(a, b), CD209 , CD235a, CD271, CD303, CD304), annexin A1, nucleoli , endoglin (CD105), ROB04, aminopeptidase N,-like 4 (DLL3) 4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H 3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, MelanA / MART1, Ras mutants, gp100, p53 mutant, proteinase 3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoint, EphA2, PAP, ML-IAP, AFP, EpCAM , ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, and gen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD 3. Fucosyl-GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYPI BI, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, N Y-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAXS, OY-T ES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAG E1, B7H3, legumain, Tie2, Page4, VEGFR2, MAD-CT- 1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.

[0167] In some embodiments, the targeting moiety capable of targeting cancer is not an antibody. Table 7 (shown in Section IV below) lists some alternative targeting moieties. Representative non-antibody targeting moieties and their corresponding antigens are shown.

[0168] In another embodiment, the targeting moiety is a protein known to be expressed on cancer cells. Such expression levels include overexpression. For example, the binding partners listed in Table 8 (shown in Section IV below) are expressed on cancer cells. The following targets can be bound:

[0169] The binding partners include the full-length or wild-type sequences of the binding partners listed in Table 8. All that is required is that the binding partner binds to the target on the cancer cell. Thus, truncated forms, analogs, etc., well known in the art, are also contemplated. , variants and derivatives.

[0170] In addition, in some embodiments, the binding partner is known to be expressed on cancer cells. The aptamer may be capable of binding to a protein that is known to bind to cancer cells. Aptamers that bind to these proteins are well known and methods for designing them are known.

[0171] Using the cell-based SELEX system, target cell characteristics are isolated from a random candidate library. A panel of specific aptamers can be selected. The antibody is dissolved in buffer, denatured, and then incubated with target cells. After washing, the antibody is bound to the target cells. The DNA or RNA that is present can be eluted by heating and then (optionally) isolated from negative cells. The supernatant may be incubated with the cells, centrifuged, and the supernatant removed. The selected sense ssDNA can be amplified using a biotin-labeled primer by Alternatively, ssARNA can be transfected with antisense biotin using streptavidin-coated beads. To improve affinity, the washing time, buffer volume, and washing time can be adjusted. The washing intensity may be increased by increasing the number of times. After the desired number of selections have been performed, The resulting ssDNA or ssRNA pool can be PCR amplified and cloned into E. coli. Shangguan et al., Aptamers evolved from live cells as effective mo lecular probes for cancer study,PNAS 103 (32:11838-11843(2006), Lyu et al. ng Cell Targeting Aptamers for Nanothera peutics Using Cell-SELEX,Theranostics 6( 9):1440-1452(2016) and Li et al., inhibition of Cell Proliferation by an An ti-EGFR Aptamer,PLoS One 6(6):e20229(201 1) See also the specific methods for designing aptamers in these references. and specific aptamers that bind to cancer cells, are hereby incorporated by reference.

[0172] For example, the aptamer may comprise SEQ ID NOs: 94-164. The aptamers may comprise SEQ ID NO: 95. These aptamers are directed against EGFR. These aptamers are provided as representative aptamers that can bind to targets displayed on cancer cells. Zhu et al., Theranostics 4(9):931-9 Other aptamers against other targets on cancer cells, such as those described in , which are also part of the present description and are incorporated by reference.

[0173] In some embodiments, the aptamers for use herein are Nanomolar to picomolar (e.g., 1 picomolar to 500 nanomolar, or K in the range of 1 picomolar to 100 nanomolar d Combine with.

[0174] 2. Targeting moieties capable of targeting tumor microenvironment cells Current cancer immunotherapy antibodies require the presence of effector cells within the tumor. However, tumors can create an immunosuppressive environment that eliminates effector cells. , resulting in less effective immunotherapy (Herbst et al. Nature. 5 15(7528):563-7(2014). Tumors that do not have a pulmonary function are sometimes called "cold tumors" (Whiteside TL et al. See Clin Cancer Res. 22(8):1845-55(2016). (I want to be).

[0175] Effector cells are non-cancer cells in the tumor stroma, specifically the tumor microenvironment, e.g., tumor-associated They can be eliminated from tumors by tumor-associated fibroblasts (TAFs) (Ziani et al., Ont Immunol. 9:414 (2018)). Non-cancer cells are sometimes called "tumor microenvironment cells." T cells in cold tumors live in the interstitium. Therefore, immunotherapeutics targeting the interface between malignant cells and the stroma may be able to overcome immune exclusion. Therefore, targeting of tumor microenvironment cells by a second targeting moiety may be possible. This can provide additional specificity to the kit or composition of the invention.

[0176] In some embodiments, the first targeting moiety binds to an antigen expressed by a cancer. and the second targeting moiety binds to an antigen expressed by cells of the tumor microenvironment. In some embodiments, the tumor microenvironment cells are fibroblasts or macrophages. These cells are tumor-associated fibroblasts or cancer-associated fibroblasts (TAFs or CAFs), or tumor-associated macrophages or cancer-associated macrophages (TAM or CAM) As used herein, "tumor associated fibroblasts" refers to cells that are present in the tumor mass. As used herein, "tumor associated" refers to fibroblasts found in or near the tumor. "Associated macrophages" refers to macrophages found in or near the tumor mass.

[0177] Figure 2 shows the targeting of one component of TWICE to cancer cells and the other to non-cancer cells in the tumor microenvironment. In this type of TWICE, the immunological binding domains of the two components are The complementary binding domains of the main and / or two components pair with each other in cancer cells and non-cancer cells. This is likely only when cancer tumor microenvironment cells are in close proximity.

[0178] TAFs are markers of activation, such as fibroblast activation protein alpha (FAP) and The expression of alpha smooth muscle actin and alpha smooth muscle actin was characterized (Barnett and Vilar, J. See Natl Cancer Inst 110(1):11-13(2018). These may create an immunosuppressive environment through direct or indirect mechanisms. The immunosuppressive effect is mediated by growth factors such as TGF-beta or cytokines, e.g., CXCL12. The indirect immunosuppressive effect may be mediated by the extracellular matrix in the tumor microenvironment. This may be mediated by remodeling of the endothelial lining (see Ziani et al., 2018).

[0179] In some embodiments, the targeting moiety of the second component is an antibody expressed by TAF. In some embodiments, the targeting moiety that targets the TAF binds to the FAP. Antibodies that bind to, such as sibrotuzumab (see US20120258119A1) FAP is expressed mainly on tumor stroma and not on normal fibroblasts ( Brennen et al.Mol Cancer Ther.11(2):257- 266(2012)).

[0180] In some embodiments, the targeting moiety of the second component is an antibody expressed by a TAM. In some embodiments, the targeting moiety that targets the TAM binds to the MAC -1 / CD11b (see EP0488061A2) or sideroflexin 3 (see WO2018020000).

[0181] D. Immune Cell Binding Domain The first immune cell binding domain and the second immune cell binding domain are bound to each other. As used herein, "immune cell" refers to a molecule that can bind to an antigen on an immune cell. An "immune cell" can be any cell involved in the immune system. Immune cells are involved in the regulation of the innate or adaptive immune response. The cells may be involved in

[0182] In some embodiments, the immune cells are natural killer cells (NK), macrophages, or In some embodiments, the T cells are γδ T cells or natural They are killer T cells (NKT cells).

[0183] 1.T cell binding domain In some embodiments, the first T cell binding domain and the second T cell binding domain are and can bind to T cells when bound to each other. In some embodiments, The first T cell binding domain and the second T cell binding domain, when bound to each other, It can bind to CD3 or the T cell receptor (TCR).

[0184] CD3 is present on all T cells and is composed of subunits designated γ, δ, ε, ζ, and η. The TCR is made up of different subunits, designated α, β, γ, and δ. Another molecule present on T cells. The cytoplasmic tail of CD3 is involved in the TCR receptor complex as well as It is sufficient to transmit the signal required for T cell activation in the absence of components. Activation of cytotoxicity begins with the interaction of the TCR with a separate cell that is itself bound to a foreign antigen. It depends on binding to major histocompatibility complex (MHC) proteins located on the outside In the absence of intervention from the immune system, only this initial TCR-MHC binding occurs, and the T cell clone A CD3-dependent signaling cascade can then occur that leads to clone proliferation, ultimately However, in some embodiments of the present invention, cytotoxicity by T cells may occur. In some cases, the first T cell binding domain and the second T cell binding domain bind to CD3 and Upon binding to CD3 and / or TCR, the CD3 and / or TCR domains mimicked immune synapse formation. Activation of cytotoxic T cells by cross-linking of the IgG1 molecule in the absence of irrelevant TCR-MHC This is because T cell cytotoxicity occurs in a clone-independent manner, i.e. This means that the cells can be activated in a manner that is independent of the specific TCR clone harbored by the cells. This is not limited to specific T cells with a particular clonal identity, but rather the entire T cell compartment. It allows the body to be rejuvenated.

[0185] In some embodiments, the first T cell binding domain is a VH domain and the second T The cell binding domain is a VL domain. In some embodiments, the first T cell binding domain The main T cell binding domain is the VL domain, and the second T cell binding domain is the VH domain. In some embodiments, the first and second T cell binding domains, when paired, form a F As used herein, "Fv" refers to an associated VH and VL. In other words, when paired, the first and second T cell binding domains form a VH and Apart from the fact that the VH and VL are not in a single chain configuration with a linker between the VH and VL. For example, it may include "scFv".

[0186] When the first and second T cell binding domains are a pair of VH and VL domains, the VH and The VL domain is specific for an antigen expressed on the surface of a T cell, e.g., CD3 or TCR. In some embodiments, the anti-CD3 or anti-TCR antibody may be a CD3 or T If the antigen is CD3, one of the possible T cell binding domains In is muromonab (muromonab-CD3 or OKT3), otelixizumab, teprinib, Derived from ipilimumab, visilizumab, foralumab, SP34 or blinatumomab It is possible. Those skilled in the art will recognize that there are a variety of anti-CD3 antibodies available, some of which are approved. It will be recognized that the therapeutic agents are or have been clinically tested in human patients ( Kuhn and Weiner Immunotherapy 8(8):889-9 06 (2016). Table 9 (shown in Section IV below) lists exemplary anti-CD Selected publications on 3 antibodies are shown. Antibodies with different isomers are well known. Table 10 (shown in Section IV below) lists exemplary antibodies. 1 shows selected publications related to TCR antibodies.

[0187] The T cell binding domain may be any antibody that is listed as a possible complementary binding domain, e.g. For example, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin mucin domain 3 (TIM-3), lymphoid Activation of lymphocytes by activating gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD 28, CD137, OX40, CD27, GITR(TNFRSF18), TIGIT, Alternatively, it may contain inducible T cell costimulatory factor (ICOS).

[0188] 2. Natural killer cell binding domain In some embodiments, the first and second immune cell binding domains are paired. When the antibody is administered, natural killer (NK) cells can be activated. In this embodiment, the first and second immune cell binding domains bind to CD16A when paired. In some embodiments, the paired immune cell binding domain and Binding to CD16A on NK cells activates NK cells against CD33-positive leukemia cells In some embodiments, the first and second immune cell binding domains are NTM-1 633 (see NCT03603665) or AFM13 (NCT01221 571).

[0189] 3. Macrophage-binding domain In some embodiments, the first and second immune cell binding domains are paired. In some embodiments, the mAb can bind to macrophages when the mAb is in a cellular state. The page is an activated macrophage.

[0190] In some embodiments, the first and second immune cell binding domains are paired. In some embodiments, the first and second The immune cell binding domain is or the entire or partial VH and / or VL of IMC-CS4 (NCT01346358) Includes parts.

[0191] In some embodiments, the first and second immune cell binding domains are paired. In some embodiments, the first and second immune cells are capable of binding to CD40. The immune cell binding domain is a whole or part of the VH and / or VL of CP-870,893. Includes.

[0192] E. Complementary Binding Domains In some embodiments, the first and second components comprise complementary binding domains. When the complementary binding domains of the two proteins pair, this leads to an additional function. The ability to induce the death of cancer cells or to increase anti-tumor immune responses, for example, An additional function may be to make the tumor microenvironment more receptive to immune cells. It can also be useful for

[0193] The two components of TWICE target cancer or the tumor microenvironment and thus bind to complementary binding domains. Additional functions mediated by in-pairing may occur in tumors or their microenvironment In some embodiments, additional functions mediated by paired complementary binding domains The activity is restricted to the tumor and its microenvironment, thereby reducing off-target effects. For example, this ability to restrict action to the tumor and its microenvironment is widely expressed and / or or target molecules having various physiological functions, such as activating or inhibiting TGF-beta. This can be an advantage in harming others.

[0194] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains can bind to the same antigen. The first complementary binding domain and the second complementary binding domain when they are bound to each other. The first immune cell-binding domain and the second immune cell-binding domain bind to the T cell. The paired complementary binding domains and the paired immune cell binding domains can bind to CD3. Both CD3- and CD4-binding may lead to more robust activation of anti-cancer immune responses.

[0195] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains can bind to different antigens on the same cell. For example, a first complementary binding domain and a second complementary binding domain when bound to each other and the first and second immune cell binding domains when bound to each other. The domains can bind to different antigens on T cells. Both the immune cell-binding domain and the binding of the two different antigens on T cells were This can lead to robust activation of immune responses.

[0196] In some embodiments, the first complementary binding domain and the second complementary binding domain when bound to each other and a complementary binding domain of the first immune cell binding domain and the second immune cell binding domain when bound to each other. The two immune cell binding domains can bind to different cells. The first complementary binding domain and the second complementary binding domain bind to the cancer cell when the first complementary binding domain and the second complementary binding domain bind to the cancer cell. The first immune cell binding domain and the second immune cell binding domain when bound to each other can combine to cause cell death. The immune cell-binding domain of 2 can bind to T cells and mediate anti-cancer immune responses. Activation of cell death pathways and anti-cancer immune responses in tumors leads to tumor regression can be enhanced.

[0197] In some embodiments, the first complementary binding domain and the second complementary binding domain are , regulating T cell, macrophage or NK cell activity when bound to each other In some embodiments, the first complementary binding domain and the second complementary binding domain The main receptors bind to costimulatory or costimulatory antigens on T cells when bound to each other. It is possible.

[0198] In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound to each other, they can induce cell death targeting cancer. In some embodiments, the first complementary binding domain and the second complementary binding domain are When bound to each other, they are capable of binding to a cell death-inducing antigen.

[0199] In some embodiments, the first complementary binding domain is a VH domain and the second complementary binding domain is a VH domain. The complementary binding domain is a VL domain. In some embodiments, the first complementary binding domain is The main is a VL domain and the second complementary binding domain is a VH domain. In some embodiments, the first and second complementary binding domains when paired are F In other words, the first and second complementary binding domains when paired may include The fragment may include an "scFv" except for the fact that the VH and VL are not in a single chain configuration. do.

[0200] In some embodiments, the VH domain of a known antibody is used as the first complementary binding domain. and the VL domain can be used as the second complementary binding domain. can.

[0201] In some embodiments, the VL domain of a known antibody is used as the first complementary binding domain. and the VH domain can be used as the second complementary binding domain. can.

[0202] 1. Complementary binding domains that regulate T cell activity In some embodiments, the first complementary binding domain and the second complementary binding domain are In some embodiments, the TCR activity can be modulated when the TCRs are bound to each other. The first complementary binding domain and the second complementary binding domain, when bound to each other, It can bind to T cell co-signaling molecules. T cell co-signaling molecules are It has been well characterized in (Chen L and Flies DB Nat. Rev Immunol 13(4):227-242(2013) , including co-stimulatory and co-inhibitory antigens. In some embodiments, co-signaling The molecule is a member of the immunoglobulin superfamily (IgSF) or tumor necrosis factor receptor superfamily. Co-inhibitory antigens are members of the immune checkpoint inhibitor family (TNFRSF). It is also sometimes called the tetramer molecule.

[0203] Table 11 (shown in Section IV below) shows that anti-tumor responses can be regulated by modulating T cell function. Examples of antibodies that can be derived and have been clinically studied are given below.

[0204] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domains are muromonab, otelixizumab, teplizumab, visilizumab, and fostatin. Contains all or part of the VH and / or VL of ralumab, SP34 or blinatumomab nothing.

[0205] In some embodiments, the first and second complementary binding domains are paired. It can sometimes bind to programmed cell death protein 1 (PD-1). In embodiments, the first and second complementary binding domains are selected from the group consisting of pembrolizumab or nivolumab. The antibody may comprise the entire or a portion of the VH and / or VL of the antibody.

[0206] In some embodiments, the first and second complementary binding domains are paired. Can occasionally bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) In some embodiments, the first and second complementary binding domains are VH of ipilimumab. and / or includes all or part of VL.

[0207] In some embodiments, the first and second complementary binding domains are paired. can bind to T cell immunoglobulin mucin domain 3 (TIM-3) In some embodiments, the first and second complementary binding domains are selected from the group consisting of TSR-022 and TSR-023. comprises all or part of the VH and / or VL of Sym023.

[0208] In some embodiments, the first and second complementary binding domains are paired. It can sometimes bind to lymphocyte activation gene 3 (LAG-3). In one embodiment, the first and second complementary binding domains are the VH and / or VH of BMS-986016. or VL.

[0209] In some embodiments, the first and second complementary binding domains are paired. Sometimes they can bind to killer cell immunoglobulin-like receptors (KIRs). In one embodiment, the first and second complementary binding domains are the VH and / or VH of lirilumab. Contains all or part of the VL.

[0210] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domain comprises all or a portion of the VH and / or VL of ceralizumab.

[0211] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second phase The complementary binding domain may be the entire or partial VH and / or VL of utomirumab or urelumab. includes some.

[0212] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domain is the VH and / or VH of PF-04518600 or BMS986178. comprises all or part of VL.

[0213] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domain comprises all or a portion of the VH and / or VL of varlilumab.

[0214] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domain is the VH and / or VL of GWN323 or BMS-986156. In whole or in part.

[0215] In some embodiments, the first and second complementary binding domains are paired. Sometimes binds to Ig and T cell immunoreceptor containing ITIM domains (TIGIT) In some embodiments, the first and second complementary binding domains are -313M32, MTIG7192A, BMS-986207 or MK-7684 V Includes all or part of H and / or VL.

[0216] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the antibody can bind to an inducible T cell costimulator (ICOS). In this embodiment, the first and second complementary binding domains are JTX-2011 (an anti-IC The VH and / or VL of the VL domain of the VL antibody (VL-OS antibody) are all or part of the VH and / or VL of the VL antibody (VL-OS antibody).

[0217] 2. Complementary binding domains that target tumor-associated macrophages (TAMs) In some embodiments, the binding of the first and second complementary binding domains to an antigen on a TAM. In combination, these antibodies mediate an increased immune response against tumors. For example, anti-CSF1R blocking antibodies Reduces AM infiltration and promotes CD8+ T cell proliferation (Ries CH et al. ancer Cell.25(6):846-59(2014)).

[0218] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The binding domains, when paired, mediate macrophage reprogramming or migration. Members of the committee can be appointed.

[0219] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second phases are capable of binding to CSF1R at certain times. The complementary binding domain is either emactuzumab / RG7155 (NCT01494688) or The whole or part of the VH and / or VL of IMC-CS4 (NCT01346358) include.

[0220] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second complementary The specific binding domain comprises all or a portion of the VH and / or VL of CP-870,893. nothing.

[0221] 3. Complementary binding domains that stimulate NK cells In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second The complementary binding domains, when paired, are capable of binding to CD16A. In some embodiments, the paired complementary binding domains bind to CD16A on NK cells. The binding activates the NK cells against CD33-positive leukemia cells. In the present invention, the first and second complementary binding domains are NTM-1633 (NCT0360366 5) or AFM13 (see NCT01221571) and / or includes all or part of VL.

[0222] 4. Complementary binding domains that inhibit checkpoint molecules expressed by cancer cells In some embodiments, the first and second complementary binding domains are paired. Sometimes, checkpoint molecules expressed by cancer cells can be inhibited. In some embodiments, the first and second complementary binding domains, when paired, It can block signals from cancer cells that inhibit immune cell activation. Several immune checkpoint molecules have been described in the literature (Park et al., 2003). experimental &Molecular Medicine 50:109(20 18), the entire disclosure of which is incorporated by reference herein for purposes of disclosure of different immune checkpoint molecules. (Incorporated herein by reference thereto).

[0223] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first and second phase The complementary binding domains are the VH and / or VH of atezolizumab, durvalumab or avelumab. or VL.

[0224] In some embodiments, the first and second complementary binding domains are paired. It can sometimes bind to CD73.

[0225] In some embodiments, the first and second complementary binding domains are CPI-006 or comprises all or part of the VH and / or VL of MEDI9447.

[0226] 5. Complementary binding domain that binds to RANK In some embodiments, the first and second complementary binding domains are paired. It can sometimes inhibit the activation of regulatory T cells.

[0227] In some embodiments, the complementary binding domains, when paired, form a RANK or binds to RANKL. RANK is expressed on cancer cells and mediates the expression of RA on regulatory T cells. It can bind to NKL and create an immunosuppressive environment (de Groot et al. .,Cancer Treatment Reviews 62:18-28(2018 In some embodiments, RANK or RANK+ expression in cancer cells is Binding to KL blocks regulatory T cell activation and stimulates the immune response.

[0228] In some embodiments, the first and second complementary binding domains are paired. Can sometimes be combined with RANK.

[0229] In some embodiments, the first and second complementary binding domains are paired. It can sometimes bind to RANKL.

[0230] In some embodiments, the first and second complementary binding domains are the VH and VH of denosumab. and / or VL.

[0231] 6. Complementary binding domain that binds to cell death-inducing antigen As used herein, a "cell death-inducing" antigen is an antigen that induces cell death after being bound to a ligand. It includes all kinds of cell death. Cell death is a programmed programmed (e.g., apoptosis) or unprogrammed (e.g., necrosis) In some embodiments, cell death is necrosis, apoptosis, or necroptosis. be.

[0232] In some embodiments, the cell death-inducing antigen comprises a cell death receptor. A member of the tumor necrosis factor receptor superfamily characterized by the presence of an intracellular "death domain" In some embodiments, the death receptor is a member of the Fas / CD95 / Apo 1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRIC K2 / KILLER, DR6 or CAR1.

[0233] In some embodiments, the first and second complementary binding domains are paired. In some embodiments, the first The first and second complementary binding domains bind to mapatumumab (anti-TRAIL-R1 / DR4 cell death inhibitor). In some embodiments, the VH and / or VL of the VL domain of a receptor antibody are included. Anti-DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER antibodies were obtained from Konatsu. tuzumab (AMG655), lexatumumab (CS1008), tigatuzumab (CS100 8) or drozitumab (PRO95780).

[0234] F. Dimerization Domain In some embodiments, the first and second components comprise a dimerization domain, as shown in FIG. As used herein, a "dimerization domain" refers to a domain that is capable of binding two proteins together. It refers to the amino acid sequence that holds the monomers (i.e., single proteins) together. Thus, A dimerization domain includes any sequence that allows two protein monomers to form a dimer. In some embodiments, the bond is covalent. , the bond is non-covalent.

[0235] The dimerization domain is linked to a complementary binding domain or an immune cell binding domain. It could be.

[0236] In some embodiments, the dimerization domain comprises a first complementary binding domain and a first immunoglobulin. and / or a second complementary binding domain and a second immune cell binding domain. Thus, dimerization promotes the association of the complementary binding domains outside the tumor site. This can reduce the likelihood that the domain will dissociate from the immune cell-binding domain.

[0237] In some embodiments, a cleavable linker (i.e., a linker that contains a cleavage site) is a first immune cell binding domain from the first complementary binding domain of the first component, and / or or mediates dissociation of the second immune cell binding domain from the second complementary binding domain. In some embodiments, the first immune cell binding domain comprises a first immune cell binding domain and and the first complementary binding domain (or both) are bound to their respective dimerization domains. until at least one cleavable linker is cleaved to release the first complementary In some embodiments, the second immune cell binding domain is , a second immune cell binding domain and / or a second complementary binding domain at least one cleavable linker such that each of the dimerization domains is released from the corresponding dimerization domain; It remains bound to the second complementary binding domain until it is cleaved.

[0238] In some embodiments, the first immune cell binding domain comprises a first dimerization domain and The first dimer is bound to the first complementary binding domain by the second dimerization domain. The somatization domain is linked to the first immune cell binding domain by a first linker, The second dimerization domain is linked to the first complementary binding domain by a second linker. and wherein the first and / or second linker is a cleavable linker.

[0239] In some embodiments, the second immune cell binding domain comprises a first dimerization domain and The first dimer is bound to a second complementary binding domain by a second dimerization domain. The somatization domain is linked to a second immune cell binding domain by a first linker, The second dimerization domain is linked to a second complementary binding domain by a second linker. and wherein the first and / or second linker is a cleavable linker.

[0240] The dimerization domain can comprise all or part of a known dimerization domain amino acid sequence. The dimerization domain contains a sequence that has homology to a known dimerization amino acid sequence. In some embodiments, the dimerization domain is , may contain amino acids optimized to improve binding.

[0241] In some embodiments, the dimerization domain is selected from the group consisting of a transcription factor or a receptor / ligand pair. Non-limiting examples of dimerization domains include the amino acid sequences of the receptor tyrosine kinase domains. Sequences from enzymes, transcription factors, 14-3-3 proteins, and G protein-coupled receptors Some examples include:

[0242] In some embodiments, the dimerization domain is a leucine zipper (coiled coil). As used herein, a "leucine zipper" includes a leucine Refers to any amino acid sequence that contains a periodic repeat of residues. Diversified leucine zippers dimerize It has been described for use in, for example, patent US 9,994,646. There are.

[0243] In some embodiments, the dimerization domain comprises an immunoglobulin domain.

[0244] In some embodiments, the dimerization domain comprises an immunoglobulin variable domain. In some embodiments, the immunoglobulin variable domain is a VH or VL domain.

[0245] In some embodiments, the dimerization domain comprises an immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain is an Fc domain. In embodiments, the immunoglobulin constant domain is a CH1 / CL, CH2, CH3 or C It is H4.

[0246] In some embodiments, the dimerization domain comprises an IgE CH2 domain.

[0247] In some embodiments, the dimerization domain comprises a TCR constant domain. The domains can be from any chain (eg, α, β, γ, or δ).

[0248] In some embodiments, the dimerization domain in the first component is a dimerization domain in the second component. For example, the dimerization domain in the first component and the dimerization domain in the second component are the same. The dimerization domain in the first and second components may be the same. In some cases, this is referred to as "homodimerization."

[0249] In some embodiments, the dimerization domain in the first component is a dimerization domain in the second component. For example, the dimerization domains in the first and second components are different from the dimerization domains in the second and third components. The domain may comprise a receptor / ligand pair. A dimerization domain in the first and second components. are different, this is sometimes referred to as "heterodimerization."

[0250] Dimers create a "knob" or "hole" that allows two proteins to dimerize. Such knob-into-hole dimerization sequences may be engineered into knob-into-hole dimerization domains. ·Into-Hole Fc or Knob-Into-Hole C H It may contain three domains. A variety of knob-into-hole examples have been described for the generation of bispecific antibodies (BIAs). See u et al., mAbs 7(1):231-242(2015) .

[0251] In some embodiments, the dimerization domain is engineered. "Engineered" means This means that the amino acid sequence has been mutated to change one or more properties of the protein. In some embodiments, the manipulation is carried out such that in the absence of the manipulation, the two protein domains communicate with each other. This electrostatically favors the association of these two domains, which would not normally occur. In some embodiments, two immunoglobulin constant domains are engineered to form an "engineered immunoglobulin" In some embodiments, the Fc region may be engineered to include a "primary IgG constant domain." results in heterodimerization of the Fc region, e.g., the CH domain.

[0252] In some embodiments, the two Fc regions are engineered to contain opposite charges and associate together. (See WO2009089004). In some embodiments, two The Fc region is engineered to contain two leucine zippers that assemble together (US9,9 In some embodiments, the two Fc regions are lysine-reactive. It is operated by configuration (see WO2017106462).

[0253] The domains are sufficiently stable that each component of TWICE is stable during production, transportation, and administration, but the T When the two components of WICE are in close proximity, the binding kinetics still remain consistent with the interaction of the immune-binding domains. and, where applicable, to favor pairing of complementary functional domains with each other. It can be done.

[0254] Several methods for generating heterodimeric Fc can be employed to generate the constructs described herein. Asymmetric IgG, i.e., two different heavy chains pair to form a heterodimer, may also be used. To create a bispecific antibody in the form of an IgG formed by y et al., one strand contains a "knob" and the other a corresponding "hole". J. B. Ridgway has engineered the CH3 domain of IgG1 by mutations that enhance its , et al. Protein Eng. 9:617-621(1196)). The two engineered heavy chains of the "hole-into-hole" approach, when co-expressed from the same cells, were homozygous for Due to steric clashes in the dimer, heterodimers will form preferentially. To facilitate heterodimer formation between chains, the chains must have similar steric clashes or repulsive charges. Using this strategy to engineer Fc domains, several other mutations have since been published (Atw ell et al.Journal of Molecular Biology 2 70:26-35(1997), Gunasekaran et al., Journal l of Biological Chemistry 285:19637-46(2 010), Moore et al.mAbs 3,546-557(2011), St. rop P et al.J.Mol.Biol.420,204-219(2012) , Von Kreudenstein et al.2013,mAbs;5:646- 54 (2013)). A combination of steric clashes and repulsive charges is used to efficiently form heterodimers. Combinations have also been employed to engineer IgG heavy chains (WO2017106462A1). Using the method described by Davis et al. (Davis et al., Protein Engineering,Design &Selection 23(4):195 -202 (2010)) engineered an asymmetric CH3 domain that enabled Fc heterodimerization. To develop a strand-exchange engineered domain (SEED) using IgG and IgA sequences, did.

[0255] G. Supplementary Functional Domains In some embodiments, the first or second component comprises a supplemental functional domain. As used herein, a "supplementary functional domain" refers to a domain that has a function when bound to a particular cell type. A complementary functional domain refers to a domain that has a function. Complementary binding domains are those that do not require pairing with another domain to have a complementary binding domain. different.

[0256] In some embodiments, the first and second components both comprise complementary functional domains. In some embodiments, only the first component comprises a supplemental functional domain.

[0257] Generally, any extracellular domain of a cell surface protein that acts as a ligand for a receptor. In some embodiments, the complementary functional domains may be used. In some embodiments, the complementary functional domain is a costimulatory molecule for T cell activation. Main is a ligand for binding to integrins.

[0258] In some embodiments, the supplemental functional domain is a member of the TGF-beta family. In some embodiments, the latent form is activated in the tumor microenvironment. The compound acts locally on tumor cells.

[0259] In some embodiments, the supplemental functional domain is a cytokine. Kine is being clinically tested in cancer (Vazquez-Lombardi et al., 2013). t al, Antibodies 2:426-451 (2013). Furthermore, by incorporating cytokines as complementary functional domains in TWICE Localized delivery of cytokines may be possible, thus improving systemic administration of cytokines In some embodiments, the cytokine is IL-2, IL-7, IL-12, IL- 15, GM-CSF, IFN-α, IFN-γ, or members of the TNF superfamily It's a bar.

[0260] In some embodiments, the supplemental functional domain is an attenuated cytokine.

[0261] As used herein, an "attenuated cytokine" refers to a cytokine that is attenuated in comparison to the wild-type cytokine. These include those with mutations that reduce their activity or those that are cut off in the tumor or tumor stromal microenvironment. A mask connected to a cytokine by a cleavable linker provides a "masking In some embodiments, the attenuated cytokine is As such, the cytokine may be a variant form of a naturally occurring cytokine. In contrast, weakened cytokines are inactive unless they are directed to target cells. Targeted attenuated cytokines may have robust antitumor activity with limited overall cardiotoxicity ( Pogue et al.,PLoS ONE 11(9):e0162472(201 6) and Pogue et al., Cytokine 1:66 (2015). (I want to be).

[0262] In some embodiments, the same unattenuated cytokines are involved in the attenuation and regulation of the immune system. Attenuated cytokines activate the immune system and promote effector function in cases where they may lead to proliferation of immune T cells. It has also been proposed to induce proliferation of receptor T cells.

[0263] In some embodiments, the attenuated cytokine is IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily In some embodiments, the attenuated cytokine is a variant form of a member of To be functional, they must be targeted to the cancer or its microenvironment.

[0264] H. Linker The inventors have proposed to include in the linker any chemical moiety that connects the components of TWICE. Can.

[0265] In some embodiments, the linker used in TWICE is a flexible linker. The linker may be a peptide, a polymer, a nucleotide, a nucleic acid, a polysaccharide, or a lipid. In some embodiments, the phosphorus The linker is a peptide linker. The length of the peptide linker is about 2 to 100, 10 to 50 or 15-30 amino acids. In some embodiments, the peptide linker is , at least 10, at least 15 or at least 20 amino acids in length and may be 80 amino acids or less, 90 amino acids or less, or 100 amino acids or less in length. In some embodiments, the linker comprises a single or repeated GGGGS (SEQ ID NO: 85 ), GGGS (SEQ ID NO: 86), GS (SEQ ID NO: 87), GSGGS (SEQ ID NO: 88), GGSG (SEQ ID NO: 89), GGSGG (SEQ ID NO: 90), GSGSG (SEQ ID NO: 91) , GSGGG (SEQ ID NO: 92), GGGSG (SEQ ID NO: 93) and / or GSSSG ( SEQ ID NO:94) sequence(s).

[0266] In some embodiments, the linker does not contain a cleavage site (i.e., a non-cleavable linker). Exemplary linkers that do not contain a cleavage site include maleimide (MPA) or SM CC linkers are an example.

[0267] In some embodiments, the linker comprises a cleavage site (ie, a cleavable linker).

[0268] In some embodiments, the linker binds the targeting moiety to the immune cell binding domain, In some embodiments, the phospholipase C is linked to a binding domain or a complementary functional domain. Car links a dimerization domain to an immune cell-binding domain or a complementary binding domain .

[0269] 1. Linker for attaching targeting moieties In some embodiments, a linker connects a targeting moiety to an immune cell binding domain. In some embodiments, the linker connects the targeting moiety to a complementary binding domain or In some embodiments, the targeting moiety is linked to a complementary functional domain. The linker connecting the binding domain or the complementary functional domain is a non-cleavable linker. In some embodiments, the targeting moiety is a complementary binding domain or a complementary The linker connecting the target functional domain is a flexible linker.

[0270] 2. Linker that connects the dimerization domains In some embodiments, a linker connects the dimerization domain to the immune cell binding domain. In some embodiments, the linker connects the dimerization domain to the complementary binding domain. Combine.

[0271] To attach the dimerization domain to the immune cell binding domain or the complementary binding domain The linker used in the above is sometimes referred to as a "dimerization domain linker." In some embodiments, the dimerization domain linker is a cleavable linker.

[0272] In some embodiments, the dimerization domain linker may include a cleavage site. In some embodiments, the dimerization domain linker comprises a protease cleavage site. In embodiments, the dimerization domain linker is a protease that can be cleaved in the tumor microenvironment. In some embodiments, the dimerization domain linker comprises one or more Contains a cleavage site for a matrix metalloprotease.

[0273] Depending on the dimerization domain employed, various dimerization domain linkers can be used in TWICE. The length of the dimerization domain linker can be used to provide flexibility for protease cleavage. However, it may be necessary to have a dimerization domain that is long enough to allow The in-linker separates the two dimerization domains too far apart to ensure dimerization. It is possible that...

[0274] In some embodiments, the first dimerization domain linker and the second dimerization domain linker In some embodiments, the first linker is the same or similar in length. The dimerization domain linker of the first dimerization domain and the second dimerization domain linker have different lengths. .

[0275] In some embodiments, the dimerization domain linker length is equal to or longer than the length of the two dimerization domains. Helps facilitate meetings.

[0276] In some embodiments, the first and second dimerization domain linkers are 5-30 In some embodiments, the first and second dimerization domain linkers are They are 8 to 16 amino acids in length.

[0277] In some embodiments, the first and second dimerization domain linkers are 5 to 15 amino acids in length. For example, a linker of this length would be suitable for a dimerization domain that is CH1 / CL. This may be appropriate in cases where

[0278] In some embodiments, the first and second dimerization domain linkers are 12 to 3 For example, a linker of this length would be suitable for a dimerization domain that is a leucine zipper In some embodiments, the first and second The dimerization domain linker is 15-20 amino acids in length. For example, The car may be adapted for certain leucine zipper dimerization domains.

[0279] Representative examples of dimerization domain linkers include SEQ ID NOs: 203 to 211. do.

[0280] I. Cleavage Site and Cleavable Linker In some embodiments, the cleavage site allows for specific cleavage at a particular position in the construct. A linker that contains a cleavage site is sometimes called a cleavable linker.

[0281] For example, the single polypeptide construct TWICE may comprise a first and a second cleavable linker. Other embodiments of this TWICE may also include a cleavable linker, e.g. For example, a dimerization domain linker may be employed.

[0282] In some embodiments, cleavage occurs by first internalizing the cell and then replicating the classical antigen. It can occur outside the cell where it is not wanted, without being integrated into a processing pathway.

[0283] In certain embodiments, at least one cleavage site is identified in an enzyme expressed by the cancer cell. Cancer cells, for example, express certain enzymes, such as proteases. By way of non-limiting example, cathepsin B is known to mediate FR, FK, Cathepsin D cleaves PRSFFRLGK (SEQ ID NO: 45); ADAM28 is composed of KPAKFFRL (SEQ ID NO: 1), DPAKFFRL (SEQ ID NO: 2), KPMKFFRL (SEQ ID NO: 3) and LPAKFFRL (SEQ ID NO: 4) were cleaved, and MMP 2 is, for example, AIPVSLR (SEQ ID NO: 46), SLPLGLWAPNFN (SEQ ID NO: 47), HPVGLLAR (SEQ ID NO: 48), GPLGVRGK (SEQ ID NO: 49) and G Cleave PLGLWAQ (SEQ ID NO: 50). Other cleavage sites listed in Table 1 or Table 2 Protease cleavage sites and cancer-associated proteases are well known in the art. Oncomine (www.oncomine.org) is a This is a database of cancer gene expression in the line, so that TWICE drugs can be used to treat cancer. For example, one skilled in the art can search the Oncomine database to find A specific protease cleavage site (or two proteases) that may be suitable for treating cancer Alternative databases include the European Bioinformatics Institute ( www.ebi.ac.uk), especially (www.ebi.ac.uk / gxa). The protease database is ExPASy Peptide Cutting r(www.ca.expasy.org / tools / peptidecutter) Examples include:

[0284] In some embodiments, the protease inhibits the expression of non-cancer cells in the tumor microenvironment, e.g., tumor It is expressed by associated macrophages or fibroblasts.

[0285] In some embodiments, the protease cleavage sites of one or more cleavable linkers are covalently linked. In some embodiments, the protein is cleaved by a localized protease. The targeting moiety may be the same as or different from the targeting moiety in the first component or the second component. and co-localized to the cancer by a targeting moiety that binds to a tumor antigen expressed by the cancer. In some embodiments, the protease is produced by cells in the tumor microenvironment. The expressed antigen is co-localized to the cancer by a targeting moiety that binds to the antigen.

[0286] The cleavage site in the cleavable linker connects the dimerization domain to the complementary binding domain and / or The cleavage site may function to release the immune cell binding domain from the unwanted cells. The complementary binding domain and / or immune cell binding domain are first and secondly expressed in the tumor microenvironment of the target cell. and / or may function to release the immune cell from the immune cell engaging domain.

[0287] In some embodiments, the dimerization domain is an immune cell binding domain or a complementary binding domain. Protease cleavage in the first and / or second cleavable linkers connecting the domains The site is a target that is expressed by the cancer or that binds to a tumor antigen expressed by the cancer. The targeting moiety is cleaved by a protease that is co-localized to the tumor. can be.

[0288] J. Linkage between complementary binding domain and immune cell binding domain In some embodiments, the first complementary binding domain is The first immune cell binding domain binds to the second immune cell as long as the first immune cell binding domain is bound. A binding portion of the first immune cell-binding domain that does not bind to the immune cell-binding domain. It is Nah.

[0289] In some embodiments, the second complementary binding domain is The second immune cell binding domain binds to the first immune cell as long as the second immune cell binding domain is bound. A binding site for the second immune cell-binding domain that does not bind to the immune cell-binding domain. It is Nah.

[0290] The complementary binding domain dissociates from the immune cell binding domain and forms a pair with the complementary domain. Upon association or dissociation of the immune cell binding domain from the complementary binding domain, pairing occurs. When this domain associates with a corresponding immune cell binding domain, this is sometimes called domain swapping. In the main exchange event, the domains move from a mismatched configuration to an active pairing configuration. .

[0291] In some embodiments, the first and second immune cell binding domains are first and second phase Fv can be formed when not bound to a complementary binding domain. In one embodiment, the first and second complementary binding domains are first and second immune cell binding domains. When not bound to the Fv, it can form an Fv.

[0292] In some embodiments, the first and second components are the immune cell binding domains of a single component. In some embodiments, the dimerization domain promotes the association of the complementary binding domain with the In this study, we investigated whether the cleavable linker in the dimerization domain linker is involved in cancer cells or their microenvironment. Cleavage of the anchor allows dissociation of the immune cell-binding domain and the complementary binding domain of a single component. In some embodiments, the first and second components do not comprise a dimerization domain.

[0293] K. Masking by an inactive binding partner In some embodiments, TWICE comprises a first and / or a second immune cell binding domain. In some embodiments, the first In some embodiments, only the immune cell binding domains of the first and second nucleotides are masked. Both immune cell binding domains of the two are masked.

[0294] For example, in some embodiments, the inactive binding partner is shown in FIG. Components of TWICE that contain additional functional domains as depicted in main section G.

[0295] In some embodiments, the first and / or second components comprising the supplemental functional domains are , including an inactive binding partner.

[0296] In some embodiments, the first inactive binding partner is a binding partner that is capable of binding to the inactive binding partner. The first immune cell-binding domain will not bind to the second immune cell-binding domain unless the first immune cell-binding domain is removed. The first immune cell-binding domain binds to the second immune cell-binding domain in a manner that does not bind to the first immune cell-binding domain.

[0297] In some embodiments, the first immune cell binding domain is a VH domain and is an inactive The binding partner is a VL domain. In some embodiments, the first immune cell binding domain The main is the VL domain and the inactive binding partner is the VH domain.

[0298] In some embodiments, a protease cleavage site is provided between the first immune cell binding domain and the first In some embodiments, the protease cleavage site separates the inactive binding partner of the The site releases the inactive binding partner from the immune cell-binding domain in the presence of a protease. It is possible to do so.

[0299] In some embodiments, the second inactive binding partner is a binding partner that is capable of binding to the inactive binding partner. The second immune cell-binding domain will not bind to the first immune cell-binding domain unless the second immune cell-binding domain is removed. The antibody binds to a second immune cell-binding domain that does not bind to the first immune cell.

[0300] In some embodiments, the second immune cell binding domain is a VH domain and is an inactive The binding partner is a VL domain. In some embodiments, the second immune cell binding domain The main is the VL domain and the inactive binding partner is the VH domain.

[0301] In some embodiments, a protease cleavage site is provided between the second immune cell binding domain and the second immune cell binding domain. In some embodiments, the protease cleavage site separates the inactive binding partner of the The site releases the inactive binding partner from the immune cell-binding domain in the presence of a protease. It is possible to do so.

[0302] In some embodiments, the protease is expressed by a cancer. In this study, proteases are expressed by cells in the tumor microenvironment, such as TAFs or TAMs. do.

[0303] In some embodiments, the protease is selected from the group consisting of (a) a first and / or second target in the agent; The targeting moiety may be the same or different and may bind to a tumor antigen expressed by the cancer. or (b) an antibody or an antigen that binds to an antigen expressed by cells in the tumor microenvironment or an antigen thereof. The targeting moiety, which is a prototypic binding fragment, is co-localized to the cancer.

[0304] L. Preparation method The various TWICEs described herein can be produced using genetic engineering techniques. The method includes expressing one or two nucleic acids in a suitable host to produce TWICE or a component thereof. For example, a nucleic acid sequence encoding all of the component moieties and the linker TWICE can be used. A vector can be made containing the sequence and the vector can be used to transform a suitable host cell. (Alternatively, each component can be expressed separately using separate vectors. good).

[0305] The nature of the host and the method of introduction of the nucleic acid into the host, as well as whether it is maintained or incorporated into an episome. Various regulatory elements may also be used in the vector, depending on whether integration is desired.

[0306] Chemical linking techniques may be employed, such as the use of maleimide or SMCC linkers.

[0307] When the targeting moiety is an aptamer, one skilled in the art can easily identify the targeting moiety by analyzing the protein, i.e., the immunoglobulin. Recognizing methods for conjugating aptamers to cell-binding domains or complementary binding domains Aptamers may be conjugated using thiol linkage or other standard conjugation chemistries. Maleimide, succinimide or SH groups can be attached to the aptamer. and linking it to the immune cell binding domain or the complementary binding domain.

[0308] II. Pharmaceutical Compositions TWICE can be employed as a pharmaceutical composition. Thus, a pharmaceutical composition is a pharma- ceutical composition that is pharma- ceutical acceptable. For example, when parenteral administration is desired, TWICE can be formulated with an acceptable carrier. Sterile, pyrogen-free water for injection or sterile, pyrogen-free saline, or TWICE may be provided in other forms acceptable for parenteral administration. It may be provided in a lyophilized form for reconstitution by addition of a liquid carrier for the bacteria. In some cases, TWICE may be provided in a single pharmaceutical composition or in two pharmaceutical compositions.

[0309] III.How to use TWICE The TWICE described herein comprises a method for treating cancer comprising administering at least a first and a second The method may be used in a patient comprising administering to the patient TWICE comprising the components of Each as described in detail in the various embodiments above.

[0310] In addition, the agents described herein are a way to direct a patient's own immune response against cancer cells. The present invention may also be used in methods comprising administering TWICE to a patient in accordance with the present invention.

[0311] In some embodiments, TWICE as described herein comprises a first targeting moiety and Targeting immune cells to cancers expressing tumor antigens that bind to both the primary and secondary targeting moieties. In some embodiments, the immune cells may be used in methods of treating These are T cells that express

[0312] In some embodiments, the TWICE described herein comprises a first target of one tumor antigen. and one tumor antigen binds to a second targeting moiety. The present invention may be used in a method for targeting immune cells to cancers expressing both tumor antigens. In some embodiments, the immune cells are CD3 or TCR expressing T cells. .

[0313] In some embodiments, the TWICE described herein is In some embodiments, the method may be used to deliver cytokines to immune cells of a patient. The first and / or second complementary functional domains of TWICE are selected from the group consisting of IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF super - Including members of the family.

[0314] In some embodiments, the patient has cancer or an identified pre-malignant condition. In some embodiments, the patient has no detectable cancer but is at high risk of developing cancer, e.g. For example, the individual has a mutation associated with an increased risk of cancer. Patients at high risk for transformation have pre-malignant tumors at high risk for transformation. In this study, patients at high risk of developing cancer had a genetic profile associated with high risk. In some embodiments, the presence of cancer or a pre-malignant condition in a patient is determined by circulating It is based on the presence of circulating tumor DNA (ctDNA) or circulating tumor cells. In some embodiments, the treatment is preemptive or prophylactic. Treatment delays or stops cancer from starting or coming back.

[0315] The amount of drug administered to the patient will be an effective amount to treat the condition in question. The first and second components of TWICE can be selected by the patient's physician as follows: They may be administered in the same formulation or in two different formulations. The compounds may be administered close enough in time to be active in the subject.

[0316] The patient undergoing treatment may be a human. The patient may be a primate or any mammal. Alternatively, the patient may be an animal, e.g., a domestic animal (e.g., a dog or cat), a laboratory animal, animals (e.g. laboratory rodents, e.g. mice, rats or rabbits), or in agriculture. The animal may be an animal of major importance, such as a horse, cow, sheep or goat.

[0317] The cancer may be a solid or non-solid malignant tumor. In some embodiments, the cancer is a solid or non-solid malignant tumor. The cancer may be any cancer, such as a solid tumor, not a lymphoma. Breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, Testicular cancer, thyroid cancer, brain tumor, esophageal cancer, stomach cancer, pancreatic cancer, colon cancer, liver cancer, leukemia, myeloma, Non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, Chronic lymphoblastic leukemia, lymphoproliferative disorders, myelodysplastic disorders, myeloproliferative disorders and pre-malignant The cancer may be a genital disease, a sexually transmitted disease, or any other cancer amenable to treatment.

[0318] In some embodiments, one of skill in the art may evaluate the tumor before initiating treatment.

[0319] For example, one skilled in the art can evaluate tumors for the presence of infiltrating T cells. In some embodiments, the method includes determining the presence of T cells in the tumor prior to initiating treatment. In some embodiments, the patient will have a solid tumor that is infiltrated with T cells. Tumor samples can be stained for T cell markers and imaging techniques can be used to identify T cells in the tumor. The presence and, optionally, density of infiltration may be indicated.

[0320] In some embodiments, the treatment method involves identifying the presence of a tumor antigen prior to initiating treatment. In some embodiments, the treatment method includes determining the concentration of a tumor antigen prior to initiating treatment. For example, one skilled in the art can assess the profile of antigens overexpressed by a tumor. It would be possible to select tumor antigens or select patients for treatment based on the Those skilled in the art will appreciate that antigen expression profiles from selected patients in which a particular antigen is overexpressed can be used to identify specific antigens. Based on the file, patients could be selected for treatment with TWICE. In some embodiments, these tumor antigens are cell surface tumor antigens. The cells may be stained for specific antigens to indicate the presence, and possibly the concentration, of tumor antigens. To determine the lower limit of expression using an in vitro T cell activation assay with TWICE These methods can be used to target TWICE to the tumor microenvironment by delivering antigens to patients. This allows researchers to confirm that the tumor has

[0321] In some embodiments, the method of treatment includes (i) determining the presence of T cells in the tumor. and (ii) determining the presence of tumor antigen(s), and / or (iii) determining the presence of tumor antigen(s). In some embodiments, the first and second targeting moieties include assessing the concentration of the original target. Expression of a tumor antigen(s) that binds to the two components of TWICE is sufficient to bring the two components of TWICE into close proximity. It is expressed at high levels in the

[0322] In some embodiments, the presence of a biomarker is used to determine whether a TWICE is present. Select patients. ut-cancer / diagnosis-staging / diagnosis / tu The mor-markers-fact-sheet is a set of markers known in the art. In some embodiments, the tumor marker is an ALK gene rearrangement or Overexpression; alpha-fetoprotein; beta-2-microglobulin; beta-human Chorionic gonadotropin; BRCA1 or BRCA2 gene mutation; BCR-ABL fusion Gene (Philadelphia chromosome); BRAF V600 mutation; C-kit / CD117 ;CA15-3 / CA27.29;CA19-9;CA-125;Calcitonin;Carcinoma fetus CEA; CD20; chromogranin A (CgA); 3, 7, 17 p21 chromosome 9; Circulating tumor cells of epithelial origin (CELLSEARCH®) ;Cytokeratin fragment 21-1;EGFR gene mutation analysis;Estrogen receptor (ER) / Progesterone receptor (PR);Fibrin / Fibrinogen;HE4;HER2 / neu gene amplification or protein overexpression; immunoglobulin; KRAS gene mutations precipitation;lactate dehydrogenase;neuron specific enolase (NSE);nuclear matrix protein 22; Programmed Death Ligand 1 (PD-L1); Prostate-specific Antigen (PSA); Thyroglobulin ;Urokinase plasminogen activator (uPA); Plasminogen activation inhibitor child (PAI-1); 5-protein signature (OVA1®); 21-gene signature (Oncotype DX®); or 70-gene signature (M Amaprint(registered trademark).

[0323] TWICE is used alone or in combination with other forms of therapy, such as surgery, radiation, conventional chemotherapy, or chemotherapy alone. The therapeutic agent may be administered in conjunction with chemotherapy or other immunotherapy.

[0324] In some embodiments, the other immunotherapy includes an immunocytokine or cytokine fusion. Cytokines are known to activate and regulate the immune system. Cytokine fusions are cell signaling proteins that are naturally made by the body in the refers to an engineered molecule that contains all or part of a cytokine. For example, a cytokine fusion The body binds all or part of the cytokine to an antibody that allows it to target the tumor. For example, Darleukin (Zegers et al. (2015) Cli n. Cancer Res., 21, 1151-60), Teleuki n (see WO2018087172).

[0325] In some embodiments, the other immunotherapy is a cancer treatment vaccination. In embodiments, cancer therapeutic vaccination enhances the body's natural defenses against cancer. have reported that IFN-γ-binding domains are expressed in 10-fold greater than IFN-γ-binding domains ... The antigens may be directed either against the IL-16 antigen or against individual mutant neoantigens.

[0326] IV. Embodiments The following numbered items refer to embodiments described herein, but are not limited to those listed herein: The embodiments are not limiting.

[0327] Item 1. A kit or composition for treating cancer in a patient, comprising a targeting a first component comprising an immune cell binding agent, said targeted immune cell binding agent being capable of targeting said cancer a first targeting moiety that binds to a tumor antigen expressed by the The immune cell-binding activity occurs when the antibody binds to a second immune cell-binding domain that is a specific target of the immune cell-binding activity. A first immune cell-binding domain, which can be either a VH domain or a VL domain, and capable of binding to a complementary antigen when bound to a second complementary binding domain. a first complementary binding domain, said second complementary binding domain being complementary to said first and when the first immune cell binding domain is a VH domain, The first complementary binding domain is a VL domain, and the first immune cell binding domain is When the first complementary binding domain is a VL domain, the first complementary binding domain is a VH domain, A first complementary binding domain binds to the first immune cell binding domain. The first immune cell binding domain binds to the second immune cell binding domain as long as the first immune cell binding domain is bound to the second immune cell binding domain. a binding partner of the first immune cell binding domain that does not bind to the first immune cell binding domain; the first component and a second component comprising a targeted immune cell binding agent, The targeted immune cell binding agent comprises a second targeting moiety, linked to the first immune cell binding domain. and when combined with said first immune cell-binding domain, said first immune cell-binding domain is capable of binding to said immune cell. and VL when the first immune cell binding domain is VH. when the second immune cell binding domain and the first complementary binding domain are bound to each other, a second complementary binding domain capable of binding to a complementary antigen, When the second immune cell binding domain is a VH domain, the second complementary binding domain is a VL domain, and the second immune cell binding domain is a VL domain, The second complementary binding domain is a VH domain, and the second complementary binding domain comprises: As long as the second immune cell binding domain is bound to the second complementary domain, such that the second immune cell binding domain does not bind to the first immune cell binding domain, comprising a second moiety that is a binding partner of the second immune cell binding domain; Kit or composition.

[0328] Item 2. The first immune cell binding domain comprises a first dimerization domain and a second dimer. a first dimerization domain that is linked to the first complementary binding domain, a first immune cell binding domain linked to said first immune cell binding domain by a first linker; The second dimerization domain is linked to the first complementary binding domain by a second linker. and wherein the first and / or second linker is a cleavable linker. The kit or composition described herein.

[0329] Item 3. The second T cell binding domain comprises a first dimerization domain and a second dimerization domain. a first dimerization domain that is bound to the second complementary binding domain via a the second T cell binding domain is linked to the second T cell binding domain by a first linker; The second dimerization domain is linked to the second complementary binding domain by a second linker. and wherein the first and / or second linker is a cleavable linker. A kit or composition according to any one of the preceding claims.

[0330] Item 4. Any of Items 2 to 3, wherein the first and second linkers are cleavable linkers. 2. The kit or composition according to claim 1.

[0331] Item 5. Any of Items 2 to 4, wherein the first linker and the second linker are the same. The kit or composition according to any one of claims 1 to 4.

[0332] Item 6. The method according to any one of Items 2 to 4, wherein the first linker and the second linker are different. A kit or composition according to any one of claims 1 to 4.

[0333] Item 7. The method according to any one of Items 2 to 6, wherein the first and second linkers have a length of 5 to 30 amino acids. A kit or composition according to any one of the preceding claims.

[0334] Item 8. The method according to any one of Items 2 to 6, wherein the length of the first and second linkers is 8 to 16 amino acids. A kit or composition according to any one of the preceding claims.

[0335] Item 9. The protease cleavage site of the first and / or second cleavable linker is The item is cleaved by proteases expressed by the cancer or tumor microenvironment cells. 9. The kit or composition according to any one of 2 to 8.

[0336] Item 10. The protease cleavage site of the first and / or second cleavable linker. is the same as the targeting moiety in at least one of the first component or the second component. or different from said antibody or an antibody that binds to a tumor antigen expressed by said cancer A protease that is co-localized to the cancer by a targeting moiety that is an antigen-binding fragment. 10. The kit or composition according to any one of items 2 to 9, wherein the nucleic acid is cleaved by

[0337] Item 11. The first and second dimerization domains are both leucine zipper, immunoglobulin, Any of items 1 to 10 that is a globulin domain or a T cell receptor (TCR) domain A kit or composition according to any one of claims 1 to 4.

[0338] Item 12. The immunoglobulin domain is an immunoglobulin variable domain or an immunoglobulin variable domain. 12. The kit or composition according to item 11, comprising a Brin constant domain.

[0339] Item 13. The immunoglobulin constant domain is CH1 / CL, CH2, CH3 or 13. The kit or composition according to item 12, comprising CH4.

[0340] Item 14. The kit of item 11, wherein the TCR domain comprises a TCR constant domain. Or a composition.

[0341] Item 15. The dimerization domain in the first component is a dimerization domain in the second component. 15. The kit or composition according to any one of items 1 to 14, wherein the kit or composition is the same as

[0342] Item 16. The dimerization domain in the first component is a dimerization domain in the second component. 15. The kit or composition according to any one of items 1 to 14, wherein the kit or composition is different from

[0343] Item 17. The first complementary binding domain and the second complementary binding domain are mutually 17. The method according to any one of items 1 to 16, wherein the compound is capable of binding to the cancer when bound to the compound. The kit or composition described herein.

[0344] Item 18. The first complementary binding domain and the second complementary binding domain are mutually When bound to the immune checkpoint molecule RANK or RANKL or 18. The kit or composition according to item 17, which is capable of binding to a cell death-inducing antigen.

[0345] Item 19. The first complementary binding domain and the second complementary binding domain are mutually complementary The antibody described in item 18 can bind to immune checkpoint molecules when bound to the antibody. The kit or composition described above.

[0346] Item 20. The first complementary binding domain and the second complementary binding domain are mutually 20. The kit or kit according to item 19, which is capable of binding to PD-L1 when bound to is a composition.

[0347] Item 21. The first and second complementary binding domains are selected from the group consisting of atezolizumab, durbarma, 21. The method according to item 20, comprising the whole or part of the VH and / or VL of ribosome or avelumab. Kit or composition.

[0348] Item 22. The first complementary binding domain and the second complementary binding domain are mutually 20. The kit according to item 19, which is capable of binding to CD73 when bound to composition.

[0349] Item 23. The first and second complementary binding domains are selected from the group consisting of CPI-006 and MEDI 23. The kit according to item 22, comprising the whole or part of VH and / or VL of 9447 or is a composition.

[0350] Item 24. The first complementary binding domain and the second complementary binding domain are mutually Item 19. The kit according to item 18, which is capable of binding to RANK when bound to composition.

[0351] Item 25. The first complementary binding domain and the second complementary binding domain are mutually Item 19. The kit according to item 18, which is capable of binding to RANKL when bound to is a composition.

[0352] Item 26. The first and second complementary binding domains are VH and / or VH of denosumab. 26. The kit or composition according to item 25, comprising all or part of a VL.

[0353] Item 27. The first complementary binding domain and the second complementary binding domain are mutually 19. The kit according to item 18, which is capable of binding to a cell death-inducing antigen when bound to Or a composition.

[0354] Item 28. The first complementary binding domain and the second complementary binding domain are mutually Fas / CD95 / Apo1, TNFR1 / p55 / CD120a when bound to , DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER, DR6, or C 28. The kit or composition according to item 27, which is capable of binding to AR1.

[0355] Item 29. The first complementary binding domain and the second complementary binding domain are mutually 28. The TRAIL-R1 / DR4-binding protein of The kit or composition described above.

[0356] Item 30. The first and second complementary binding domains are VH and / or VH of mapatumumab. 30. The kit or composition of item 29, wherein said VL comprises all or part of said VL.

[0357] Item 31. The first complementary binding domain and the second complementary binding domain are mutually DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLE when bound to 29. The kit or composition according to item 28, capable of binding to R.

[0358] Item 32. The first and second complementary binding domains are conatumumab (AMG655). , lexatumumab, tigatuzumab (CS1008) or drozitumab (PRO9578 32. The kit or composition according to item 31, comprising:

[0359] Item 33. The first complementary binding domain and the second complementary binding domain are mutually When bound to the extracellular matrix, it can bind to molecules associated with the extracellular matrix. The kit or composition according to any one of items 1 to 16.

[0360] Item 34. The first complementary binding domain and the second complementary binding domain are mutually When bound to the IL-1 receptor, it binds to T cells, macrophages, or natural killer cells. 17. The kit or composition according to any one of items 1 to 16,

[0361] Item 35. The first complementary binding domain and the second complementary binding domain are mutually 35. The kit or composition according to item 34, which is capable of binding to T cells when bound to Composition.

[0362] Item 36. The first complementary binding domain and the second complementary binding domain are each When bound to CD3, programmed cell death protein 1 (PD-1), cytotoxicity T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer cell immunoglobulin like receptor (KIR), CD28, CD137, OX40, CD27, GITR (TNFR SF18), TIGIT or inducible T cell costimulator (ICOS). Item 36. The kit or composition according to Item 35,

[0363] Item 37. The first and second complementary binding domains bind to CD3 37. The kit or composition according to item 36, which is capable of binding to

[0364] Item 38. The first and second complementary binding domains are selected from the group consisting of muromonab, otelixizumab, , teplizumab, visilizumab, foralumab, SP34 or blinatumomab VH and 38. The kit or composition according to item 37, comprising the whole or part of a VL and / or a VL.

[0365] Item 39. The first and second complementary binding domains bind to each other to form a PD- 37. The kit or composition according to item 36, which is capable of binding to 1.

[0366] Item 40. The first and second complementary binding domains are selected from the group consisting of pembrolizumab and niborin. 40. A kit or composition according to item 39, comprising all or part of the VH and / or VL of a mab. Composition.

[0367] Item 41. The first and second complementary binding domains bind to each other to form a CTL. 37. The kit or composition according to item 36, capable of binding to A-4.

[0368] Item 42. The first and second complementary binding domains are VH and / or VH of ipilimumab. 42. The kit or composition according to item 41, wherein said VL comprises all or part of said VL.

[0369] Item 43. The first and second complementary binding domains, when bound to each other, form a TIM 37. The kit or composition according to item 36, capable of binding to -3.

[0370] Item 44. The first and second complementary binding domains are selected from the group consisting of TSR-022 and Sym0 44. The kit or composition according to item 43, comprising all or part of VH and / or VL of 23. Composition.

[0371] Item 45. The first and second complementary binding domains, when bound to each other, form a LAG 37. The kit or composition according to item 36, capable of binding to -3.

[0372] Item 46. The first and second complementary binding domains are VH and VH of BMS-986016. 46. ​​The kit or composition according to item 45, comprising a whole or part of a VL and / or a VL.

[0373] Item 47. The first and second complementary binding domains bind to each other to form a KIR 37. The kit or composition according to item 36, which is capable of binding to

[0374] Item 48. The first and second complementary binding domains are VH and / or VH of lirilumab. 48. The kit or composition according to item 47, comprising all or part of a VL.

[0375] Item 49. The first and second complementary binding domains bind to CD2 37. The kit or composition according to item 36, which is capable of binding to 8.

[0376] Item 50. The first and second complementary binding domains are VH and / or VH of ceralizumab. 50. The kit or composition of item 49, wherein said VL comprises all or part of said VL.

[0377] Item 51. The first and second complementary binding domains bind to CD1 37. The kit or composition according to item 36, which is capable of binding to

[0378] Item 52. The first and second complementary binding domains are utomirumab or urelumab. 52. The kit or composition according to item 51, comprising the whole or part of the VH and / or VL of .

[0379] Item 53. The first and second complementary binding domains bind to OX4 37. The kit or composition according to item 36, wherein the kit or composition is capable of binding to 0.

[0380] Item 54. The first and second complementary binding domains are selected from the group consisting of PF-04518600 and PF-04518600. 54. The method according to item 53, comprising the whole or part of the VH and / or VL of BMS986178. Kit or composition.

[0381] Item 55. The first and second complementary binding domains bind to CD2 37. The kit or composition according to item 36, which is capable of binding to .

[0382] Item 56. The first and second complementary binding domains are VH and / or VH of varlilumab. 56. The kit or composition of item 55, wherein said VL comprises all or part of said VL.

[0383] Item 57. The first and second complementary binding domains, when bound to each other, form a GIT 37. The kit or composition according to item 36, which is capable of binding to R(TNFRSF18). .

[0384] Item 58. The first and second complementary binding domains are selected from the group consisting of GWN323 and BMS-9. 58. The kit or kits according to item 57, comprising the whole or part of the VH and / or VL of 86156. Or compositions.

[0385] Item 59. The first and second complementary binding domains bind to each other to form a TIG 37. The kit or composition according to item 36, capable of binding to an IT.

[0386] Item 60. The first and second complementary binding domains are selected from the group consisting of OMP-313M32, MTI The entire VH and / or VL of G7192A, BMS-986207 or MK-7684 60. The kit or composition according to item 59, comprising a body or part thereof.

[0387] Item 61. The first and second complementary binding domains, when bound to each other, form an ICO 37. The kit or composition according to item 36, capable of binding to S.

[0388] Item 62. The first and second complementary binding domains are VH and / or VH of JTX-2011. 62. The kit or composition according to item 61, comprising all or a part of a VL.

[0389] Item 63. The first complementary binding domain and the second complementary binding domain are mutually 35. The kit according to item 34, which is capable of binding to macrophages when bound to Or a composition.

[0390] Item 64. The first complementary binding domain and the second complementary binding domain are mutually Item 64. The kit or kit according to item 63, which is capable of binding to CSF1R when bound to is a composition.

[0391] Item 65. The first and second complementary binding domains are selected from the group consisting of emacutuzumab and IMC- 66. The kit according to item 65, comprising the whole or part of the VH and / or VL of CS4, composition.

[0392] Item 66. The first complementary binding domain and the second complementary binding domain are each 64. The kit according to item 63, which is capable of binding to CD40 when bound to composition.

[0393] Item 67. The first and second complementary binding domains are VH and VH of CP-870,893. 67. The kit or composition according to item 66, comprising a whole or part of a VL and / or a VL.

[0394] Item 68. The first complementary binding domain and the second complementary binding domain are mutually 35. The method according to claim 34, which is capable of binding to natural killer cells when bound to Kit or composition.

[0395] Item 69. The first complementary binding domain and the second complementary binding domain are mutually 69. The kit according to item 68, which is capable of binding to CD16A when bound to is a composition.

[0396] Item 70. The first and second complementary binding domains are NTM-1633 or AFM 69. A kit or a composition according to Item 69, comprising all or part of VH and / or VL of 13. Composition.

[0397] Item 71. The first complementary binding domain and the second complementary binding domain when bound to each other. and a specific binding domain, and the first immune cell binding domain and the specific binding domain when bound to each other. Any of items 1 to 70, wherein the second immune cell binding domain is capable of binding to the same antigen. The kit or composition according to any one of claims 1 to 4.

[0398] Item 72. The first complementary binding domain and the second complementary binding domain when bound to each other. and a specific binding domain, and the first immune cell binding domain and the specific binding domain when bound to each other. The second immune cell binding domain can bind to a different antigen on the same cell. 71. The kit or composition according to any one of items 1 to 70.

[0399] Item 73. The first complementary binding domain and the second complementary binding domain when bound to each other. and a specific binding domain, and the first immune cell binding domain and the specific binding domain when bound to each other. Any of items 1 to 70, wherein the second immune cell binding domain is capable of binding to a different cell. A kit or composition according to any one of claims 1 to 4.

[0400] Item 74. The first and second immune cell binding domains are complementary to the first and second immune cell binding domains. Any of items 1 to 73, which is capable of forming an Fv when not bound to the binding domain. 2. The kit or composition according to claim 1.

[0401] Item 75. The first and second complementary binding domains bind to the first and second immune cells. Any of items 1 to 74, which is capable of forming an Fv when not bound to the binding domain. 2. The kit or composition according to claim 1.

[0402] Item 76. A kit or composition for treating cancer in a patient, comprising a targeting a first component comprising a targeted immune cell binding agent, said targeted immune cell binding agent being a first targeting moiety that binds to a tumor antigen expressed by the cancer, The immune cell-binding activity occurs when the antibody binds to a second immune cell-binding domain that is not a target of the immune cell binding activity. a first immune cell binding domain, which may be either a VH domain or a VL domain, a first inactive binding partner of the first immune cell binding domain, The first immune cell binding domain binds to the second immune cell binding domain unless the active binding partner is removed. and a second immune cell-binding domain that does not bind to the first immune cell-binding domain. and when the first immune cell-binding domain is a VH domain, and when the first immune cell binding domain is a VL domain, the VH domain is the first inactive binding partner, which is the main; a protease cleavage site separating the first inactive binding partner from the cancer or expressed by cells of the tumor microenvironment, or (a) said first and second and / or a second targeting moiety that may be the same or different as the targeting moiety caused by said cancer. or (b) an antigen expressed by cells in the tumor microenvironment. and colocalizing the cancer with a targeting moiety that is an antibody or antigen-binding fragment thereof that binds to In the presence of a protease that is localized in the immune cell, the immune cell-binding domain is freed from the unbinding domain. the protease cleavage site capable of releasing an active binding partner; and a first component comprising a first complementary functional domain capable of cell binding; and a second component comprising a targeted immune cell binding agent, The agent comprises a second targeting moiety, a second immune cell binding domain, and optionally, an immune cell binding domain. The second component comprises a second complementary functional domain capable of binding to the first component. The kit or composition.

[0403] Item 77. The kit or method according to item 76, wherein the second component comprises a supplemental functional domain. Or compositions.

[0404] Item 78. The complementary functional domains of the first and / or second components are of a receptor. 78. The kit or composition according to any one of items 76 to 77, comprising a ligand.

[0405] Item 79. The complementary functional domain is a potential member of the TGF-beta family. 80. The kit or composition according to item 78, in the form of

[0406] Item 80. The complementary functional domains of the first and / or second components are 80. The kit or composition according to item 78, comprising

[0407] Item 81. The cytokine is IL-2, IL-7, IL-12, IL-15, GM - A member of the CSF, IFN-α, IFN-γ, or TNF superfamily 79. The kit or composition according to item 78.

[0408] Item 82. The complementary functional domain of the first and / or second component is a weakened side 81. The kit or composition according to item 80, comprising tocainide.

[0409] Item 83. The weakened cytokine is IL-2, IL-7, IL-12, IL-15 , GM-CSF, IFN-α, IFN-γ or members of the TNF superfamily 83. The kit or composition of item 82, which is a variant.

[0410] Item 84. The second component is a second inactive binding portion of the second immune cell binding domain. The toner further comprises a second inert binding partner, The second immune cell binding domain binds to the first immune cell binding domain unless removed. and a second immune cell-binding domain that binds to the second immune cell-binding domain but does not bind to the second immune cell-binding domain. wherein the second immune cell binding domain is a VH domain and the inactive binding partner wherein said first immune cell binding domain is a VL domain and said second immune cell binding domain is a VL domain. The inactive binding partner is a VH domain, and further comprises a protease cleavage site in front of the VH domain. a second immune cell binding domain separating said second immune cell binding domain from said second inert binding partner; The protease cleavage site is expressed by the cancer, or (a) in the drug. the first and / or second targeting moieties may be the same or different and may be or (b) binds to a tumor antigen expressed by a cell in the tumor microenvironment. The targeting moiety is an antibody or antigen-binding fragment thereof that binds to the expressed antigen. the immune cell binding domain in the presence of a protease that is co-localized to a cancer 84. Any one of items 76 to 83, which can liberate the inactive binding partner from Item 3. The kit or composition according to item 1.

[0411] Item 85. The method according to items 1 to 8, wherein the first component is not covalently bonded to the second component. 5. A kit or composition according to any one of claims 4.

[0412] Item 86. The first component is covalently bonded to the second component, any one of items 1 to 85. A molecule comprising the kit or composition described in any one of claims 1 to 4.

[0413] Item 87. The first component is covalently linked to the second component via a cleavable linker. 87. The single molecule according to item 86,

[0414] Item 88. The first immune cell binding domain and the second immune cell binding domain are When bound to each other, they bind to T cells, macrophages, or natural killer cells. 88. The kit or composition according to any one of items 1 to 87,

[0415] Item 89. The first immune cell binding domain and the second immune cell binding domain are 89. The kit according to item 88, which is capable of binding to T cells when bound to each other. is a composition.

[0416] Item 90. The first immune cell binding domain and the second immune cell binding domain are When bound to each other, CD3, T cell receptor, and programmed cell death protein 1 (PD -1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin T-cell mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), killer Cellular immunoglobulin-like receptors (KIR), CD28, CD137, OX40, CD27, GITR (TNFRSF18), TIGIT or inducible T cell costimulatory factor (ICOS) 90. The kit or composition according to item 89, which is capable of binding to

[0417] Item 91. The first immune cell binding domain and the second immune cell binding domain are each 91. The kit or composition according to item 90, which is capable of binding to CD3 when bound to Composition.

[0418] Item 92. The first and second immune cell binding domains are selected from the group consisting of muromonab, otelixisumab, and otelixisumab. VH of blinatumomab, teplizumab, visilizumab, foralumab, SP34 or blinatumomab and / or a whole or part of a VL.

[0419] Item 93. The first immune cell binding domain and the second immune cell binding domain are 91. The kit according to item 90, which is capable of binding to a T cell receptor when bound to each other. or composition.

[0420] Item 94. The first and second immune cell binding domains bind to each other to form a PD 91. The kit or composition according to item 90, capable of binding to -1.

[0421] Item 95. The first and second immune cell binding domains are selected from the group consisting of pembrolizumab and nivolumab. 95. The kit according to item 94, comprising the whole or part of the VH and / or VL of lumab, composition.

[0422] Item 96. The first and second immune cell binding domains, when bound to each other, produce a CT 91. The kit or composition according to item 90, which is capable of binding to LA-4.

[0423] Item 97. The first and second immune cell binding domains are VH and / or VH of ipilimumab. 97. The kit or composition of item 96, comprising a whole or part of a VL or VL.

[0424] Item 98. The first and second immune cell binding domains, when bound to each other, form a TI. 91. The kit or composition according to item 90, capable of binding to M-3.

[0425] Item 99. The first and second immune cell binding domains are selected from the group consisting of TSR-022 and Sym. 99. The kit according to item 98, comprising the whole or part of the VH and / or VL of 023, composition.

[0426] Item 100. The first and second immune cell binding domains, when bound to each other, form a L 91. The kit or composition according to item 90, which is capable of binding to AG-3.

[0427] Item 101. The first and second immune cell binding domains are V of BMS-986016 101. The kit or composition according to item 100, comprising all or part of H and / or VL.

[0428] Item 102. The first and second immune cell binding domains, when bound to each other, 91. The kit or composition according to item 90, which is capable of binding to IR.

[0429] Item 103. The first and second immune cell binding domains are VH and / or VH of lirilumab. 103. The kit or composition according to item 102, comprising a whole or part of a VL or VL.

[0430] Item 104. The first and second immune cell binding domains, when bound to each other, form a C 91. The kit or composition according to item 90, capable of binding to D28.

[0431] Item 105. The first and second immune cell binding domains are VH and / or VH of ceralizumab. or the whole or part of the VL.

[0432] Item 106. The first and second immune cell binding domains, when bound to each other, form a C 91. The kit or composition according to item 90, which is capable of binding to D137.

[0433] Item 107. The first and second immune cell binding domains are utomirumab or urelamma. 107. A kit according to item 106, comprising all or part of the VH and / or VL of a mab, composition.

[0434] Item 108. The first and second immune cell binding domains, when bound to each other, 91. The kit or composition according to item 90, capable of binding to X40.

[0435] Item 109. The first and second immune cell binding domains are selected from the group consisting of PF-04518600 and PF-04518600. or BMS986178, including all or a part of the VH and / or VL of BMS986178, The kit or composition described herein.

[0436] Item 110. The first and second immune cell binding domains, when bound to each other, form a C 91. The kit or composition according to item 90, which is capable of binding to D27.

[0437] Item 111. The first and second immune cell binding domains are VH and / or VH of varlilumab. or the whole or part of the VL.

[0438] Item 112. The first and second immune cell binding domains, when bound to each other, form a G Item 90, a kit or composition according to Item 90, capable of binding to ITR (TNFRSF18). Composition.

[0439] Item 113. The first and second immune cell binding domains are selected from the group consisting of GWN323 and BMS 113. The peptide according to item 112, comprising the whole or part of VH and / or VL of -986156. or composition.

[0440] Item 114. The first and second immune cell binding domains bind to each other to form a T 91. The kit or composition according to item 90, which is capable of binding to IGIT.

[0441] Item 115. The first and second immune cell binding domains are selected from the group consisting of OMP-313M32, M VH and / or VL of TIG7192A, BMS-986207 or MK-7684 15. The kit or composition according to item 114, comprising in whole or in part:

[0442] Item 116. The first and second immune cell binding domains, when bound to each other, form an I 91. The kit or composition according to item 90, capable of binding to COS.

[0443] Item 117. The first and second immune cell binding domains are VH and VH of JTX-2011. 117. The kit or composition according to item 116, comprising a whole or part of a VL and / or a VL.

[0444] Item 118. The first immune cell binding domain and the second immune cell binding domain 89. The method according to claim 88, which is capable of binding to macrophages when bound to each other. Kit or composition.

[0445] Item 119. The first immune cell binding domain and the second immune cell binding domain 119. The peptide according to item 118, which is capable of binding to CSF1R when bound to each other. or composition.

[0446] Item 120. The first and second immune cell binding domains are selected from the group consisting of emactuzumab and IM 119. The kit according to item 119, comprising the whole or part of the VH and / or VL of C-CS4. Or a composition.

[0447] Item 121. The first immune cell binding domain and the second immune cell binding domain 121. The kit according to item 120, which is capable of binding to CD40 when bound to each other. or composition.

[0448] Item 122. The first and second immune cell binding domains are V of CP-870,893 122. The kit or composition according to item 121, comprising all or part of H and / or VL.

[0449] Item 123. The first immune cell binding domain and the second immune cell binding domain , which can bind to natural killer cells when bound to each other, The kit or composition described herein.

[0450] Item 124. The first immune cell binding domain and the second immune cell binding domain 124. The protein according to item 123, which is capable of binding to CD16A when bound to each other. or composition.

[0451] Item 125. The first and second immune cell binding domains are selected from the group consisting of NTM-1633 and A 125. The kit or kits according to item 124, comprising the whole or part of the VH and / or VL of FM13. Or compositions.

[0452] Item 126. The first targeting moiety and the second targeting moiety are different. 126. The kit or composition according to any one of items 1 to 125.

[0453] Item 127. The first targeting moiety and the second targeting moiety are the same. 126. The kit or composition according to any one of items 1 to 125.

[0454] Item 128. The first and / or second targeting moiety is an antibody or an antigen-binding 128. The kit or composition according to any one of items 1 to 127, comprising a functional fragment.

[0455] Item 129. The first and / or second targeting moiety is a DNA aptamer, a RNA aptamer, a A aptamer, albumin, lipocalin, fibronectin, ankyrin, finomer, Obody, DARPin, knotin, avimer, atrimer, an Anti-callin, affilin, affibody, bicyclic peptide, c ys-knot, FN3 (adnectin, centryrins, pro nectin or TN3), or any of items 1 to 128 containing a Kunitz-type domain The kit or composition according to any one of claims 1 to 4.

[0456] Item 130. The second targeting moiety binds to a tumor antigen expressed by the cancer. 130. The kit or composition according to any one of items 1 to 129.

[0457] Item 131. The first and / or second targeting moiety is an α4 integrin, A3 3, ACVRL1 / ALK1, ADAM17, ALK, APRIL, BCMA, C242 , CA125, cadherin-19, CAIX, CanAg, carbonic anhydrase IX, CCN1 , CCR4, CD123, CD133, CD137 (4-1BB), CD138 / Cindel Can1, CD19, CD2, CD20, CD22, CD30, CD33, CD37, CD 38, CD4, CD40, CD44, CD45, CD48, CD5, CD52, CD56 , CD59, CD70, CD70b, CD71, CD74, CD79b, CD80, CD 86, CD98, CEA, CEACAM, CEACAM1, CK8, c-Kit, CLD N1, CLDN18, CLDN18.2, CLDN6, c-met / HGFR, c-RE T, Cripto, CTLA-4, CXCR4, DKK-1, DLL3, DLL4, TR AIL-R2 / DR5, DRS, EGFL7, EGFR, EGFRvIII, Endogly , ENPP3, EpCAM, EphA2, episialin, FAP, FGFR1, FGF R2, FGFR3, FGFR4, fibronectin extra domain B, FLT-3, flt4, folate receptor 1, GCC, GD2, GD3, glypican-3, glypican, GM 3, GPNMB, GPR49, GRP78, Her2 / Neu, HER3 / ERBB3, HLA-DR, ICAM-1, IGF-1R, IGFR, IL-3Ra, integrin α 5β1, integrin α6β4, integrin αV, integrin αVβ3, Lewis Y, Lewis y / b antigen, LFL2, LIV-1, Ly6E, MCP-1, mesothelin, MMP -9, MUC1, MUC18, MUC5A, MUC5AC, myostatin, NaPi2b , Neuropilin 1, NGcGM3, NRP1, P-cadherin, PCLA, PD-1, PDGFRa, PD-L1, PD-L2, phosphatidylserine, PIVKA-II, P LVAP, PRLR, progastrin, PSCA, PSMA, RANKL, RG1, Si glec-15, SLAMF6, SLAMF7, SLC44A4, STEAP-1, TA CSTD-2, tenascin-C, TPBG, TRAIL-R1 / DR4, TROP-2, T WEAKR, TYRP1, VANGL2, VEGF, VEGF-C, VEGFR-2, 128. The method according to claim 127, further comprising administering to said patient an antibody or an antigen-binding fragment thereof which binds to VEGF-R2. The kit or composition described above.

[0458] Item 132. The first and / or second targeting moiety is an anti-alpha4 integrin antibody. , anti-CD137 antibody, anti-CCR4 antibody, anti-CD123 antibody, anti-CD133 antibody, anti-CD1 38 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD 38 antibody, anti-CD40 antibody, anti-CD49d antibody, anti-CD52 antibody, anti-CD70 antibody, anti-C D74 antibody, anti-CD79b antibody, anti-CD80 antibody, anti-CEA antibody, anti-cMet antibody, anti-C ripto antibody, anti-CTLA-4 antibody, anti-DLL3 antibody, anti-TRAIL-2 / DR5 antibody , anti-E-cadherin antibody, anti-endoglin antibody, anti-EpCAM antibody, anti-epidermal growth factor receptor antibody Antibody, anti-FGFR3 antibody, anti-fibronectin extra domain B antibody, anti-folate receptor body 1 antibody, anti-glypican 3 antibody, anti-gp95 / 97 antibody, anti-Her2 antibody, anti-IGF-1 R antibody, anti-IL-13R antibody, anti-IL-4 antibody, anti-IL-6 antibody, anti-MMP-9 antibody, anti- MUC1 antibody, anti-mucin core protein antibody, anti-NGcGM3 antibody, anti-P-cadherin antibody body, anti-PD-L1 antibody, anti-p-glycoprotein antibody, anti-PSCA antibody, anti-PSMA antibody, anti- SLAMF7 antibody, anti-TRAIL-R1 / DR4 antibody, anti-transferrin antibody, anti-TR The present invention relates to an antibody, or an antigen-binding fragment thereof, which is an OP-2 antibody or an anti-VEGF antibody. 128. A kit or composition according to item 128.

[0459] Item 133. The first and / or second targeting moiety is selected from the group consisting of alemtuzumab, ande Caliximab, atezolizumab, avelumab, BCD-100, bevacizumab, BGB- A317, blinatumomab, brentuximab, BU59, camrelizumab, carotid Mab, catumaxomab, cemiplimab, cetuximab, daratumumab, depatuxizumab , dinutuximab, DS-8201, durvalumab, edrecolomab, elotuzumab, G544, gemtuzumab, glembatumumab, GP1.4, hp67.6, IBI308 , Ibritumomab, Inotuzumab, Ipilimumab, Isatuximab, L19IL2, L1 9TNF, margetuximab, mirvetuximab, mogamulizumab, moxetumomab, na Talizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olalutumumab oportuzumab, panitumumab, PDR001, pembrolizumab, pertuzumab, Polatuzumab, racotumomab, ramucirumab, rituximab, rovalpituzumab, sacitus Zumab, SM3, TAK-164, tositumomab, trastuzumab, tremelimumab, Blituximab, urelumab, utomilumab, XMAB-5574, or zolbetuximab 129. The kit or composition according to item 128, comprising a mab.

[0460] Item 134. The first and / or second targeting moiety is selected from the group consisting of IL-2, IL-4, I L-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL-13 , stem cell factor, insulin-like growth factor (IGF), or CD40, items 1 to 12 8. A kit or composition according to any one of claims 7.

[0461] Item 135. The first and / or second targeting moiety is selected from the group consisting of IL-2, IL-4, I L-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL-13 , stem cell factor, insulin-like growth factor (IGF), or CD40 full-length sequence, 134. A kit or composition according to any one of claims 1 to 134.

[0462] Item 136. The first and / or second targeting moiety is selected from the group consisting of IL-2, IL-4, I L-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL-13 , stem cell factor, insulin-like growth factor (IGF) or truncated forms, analogs, and / or markers of CD40 135. The kit or composition according to item 134, comprising a rhamnoside or derivative thereof.

[0463] Item 137. The first and / or second targeting moiety is an IL-2 receptor, an IL- 4. IL-6, melanocyte-stimulating hormone receptor (MSH receptor), transferrin receptor receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EG F receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR or CD4 135. The kit or composition according to any one of items 1 to 134, wherein the kit or composition binds to IL-136.

[0464] Item 138. The second targeting moiety is directed to an antigen expressed by tumor microenvironment cells. 130. The kit or composition according to any one of items 1 to 129, which binds.

[0465] Item 139. The tumor microenvironment cells are fibroblasts or macrophages. 39. The kit or composition according to claim 38.

[0466] Item 140. The antigen expressed by fibroblasts is a fibroblast activation protein. Item 140. The kit or composition according to item 139,

[0467] Item 141. Antigens expressed by macrophages are MAC-1 / CD11b or Item 140. The kit or composition according to item 139, wherein the compound is sideroflexin 3.

[0468] Item 142. The first targeting moiety and / or the second targeting moiety in a patient. A method for treating a cancer expressing a tumor antigen that binds to the antibody, comprising the steps of: The method comprises administering to the patient a composition described in any one of claims.

[0469] Item 143. Prior to administering the composition, the cancer is evaluated for the presence of infiltrating immune cells. Item 143. The method according to Item 142.

[0470] Item 144. Prior to administering the composition, the cancer is evaluated for the presence of a tumor antigen. The method according to any one of Items 142 to 143.

[0471] Item 145. The first targeting moiety and the second targeting moiety bind to the same antigen. The method according to any one of Items 142 to 144,

[0472] Item 146. The first targeting moiety and the second targeting moiety bind to different antigens. The method according to any one of Items 142 to 144,

[0473] Item 147. A tumor antigen that binds to the first and / or second targeting moiety is expressed. The cancers for which the present invention is being studied are breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, and melanoma. , lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, stomach cancer, pancreatic cancer, colon cancer, liver cancer , leukemia, myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia Myelodysplastic disorders, myelodysplastic disorders, myeloproliferative disorders, myelodysplastic disorders, myeloblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorders, myelodysplastic disorders, myelodysplastic disorders, myeloblastic leukemia ...odysplastic disorders, myel Any one of items 142 to 146, which is a proliferative disease or a premalignant disease. 2. The method according to claim 1.

[0474] Item 148. A method for the preparation of a method for the preparation of a subject comprising administering to said subject an antibody that binds both the first and / or second targeting moiety in said subject. A method for targeting immune cells to a cancer expressing a tumor antigen comprising the steps of: The method comprising administering to the patient a composition described in any one of claims.

[0475] Item 149. A tumor antigen in a patient that binds to the first targeting moiety. and one tumor antigen is linked to the second targeting moiety. A method for targeting immune cells to a cancer in which an allergen is expressed, comprising any one of items 1 to 141. The method comprises administering to the patient the composition of claim 1.

[0476] Item 150. A method for delivering cytokines to immune cells of a patient, comprising the steps of: 7 to a patient, The first and / or second complementary functional domains are selected from the group consisting of IL-2, IL-7, IL-12, IL- -15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily members The method further comprises:

[0477] V. Supplementary Table The following supplemental tables are referenced in the above application:

[0478] [Table 2-1]

[0479]

Table 2-2

[0480]

Table 3-1

[0481]

Table 3-2

[0482]

Table 3-3

[0483]

Table 4-1

[0484]

Table 4-2

[0485]

Table 5

[0486]

Table 6-1

[0487]

Table 6-2

[0488]

Table 6-3

[0489]

Table 6-4

[0490]

Table 6-5

[0491]

Table 6-6

[0492]

Table 6-7

[0493]

Table 6-8

[0494]

Table 6-9

[0495]

Table 6-10

[0496]

Table 6-11

[0497]

Table 6-12

[0498]

Table 6-13

[0499] [Table 7]

[0500] [Table 8]

[0501] [Table 9-1]

[0502] [Table 9-2]

[0503] [Table 10-1]

[0504] [Table 10-2]

[0505] [Table 11] EXAMPLES

[0506] Example 1. TWICE blocks two inhibitory pathways in immune cells TWICEs that act on various cancers and immune cells can be developed. These examples are particularly This is to help illustrate how one skilled in the art can create a TWICE with a given functionality.

[0507] For example, TWICE, which contains a synergistic combination of two checkpoint inhibitor antibodies, was created. It can be done.

[0508] To begin with, one of skill in the art would first select a targeting moiety that is appropriate for a given cancer. Targeting moieties are selected based on the literature regarding the predominance of certain antigens for a given cancer type. Alternatively, antigens expressed by the cancer in a particular patient may be evaluated. For example, rituximab (an anti-CD20 antibody) is used to target CD20-positive lymphomas. Cetuximab (an anti-EGFR antibody) can also be used to treat certain solid tumors. can be targeted.

[0509] TWICE may allow the use of two targeting moieties: a first component and It is also possible for one of skill in the art to select two targeting moieties to target both the first and second components. It is possible. CAI25 is used to target one component and β-actin is used to target the other component. EpCAM can be used to target ovarian cancer.

[0510] After selecting an appropriate targeting moiety, one of skill in the art can then select any type of immune cell binding domain. and determining which complementary binding domains may have the greatest therapeutic effect when paired. It is also possible.

[0511] Figure 1 shows how TWICE employs two targeting domains against cancer cells. We show how these proteins can be used to enable activation via two signaling pathways in immune cells.

[0512] TWICE inhibits two checkpoint molecules to enhance antitumor immune responses This results in the production of a tumor-specific checkpoint complex that inhibits the anti-cancer immune response. There is a possibility that the benefits will be outweighed by the harm.

[0513] For example, the VH of ipilimumab (anti-CTLA4 antibody) as an immune cell binding domain, The first component comprises the VL of nivolumab (anti-PD-1 antibody) as a complementary binding domain. It is possible to create a TWICE that can be expressed as , the VL of ipilimumab (anti-CTLA4 antibody) as an immune cell binding domain, and the complementary It may contain the VH of nivolumab (anti-PD-1 antibody) as a binding domain. The function of ICE is to recognize either component as long as one component has a VH and the other has a VL. has the VH or VL of a given antibody.

[0514] By producing TWICE in this manner, the first and second components of TWICE At the cancer site, both the VH of ipilimumab and The VL, and the VH and VL of nivolumab are pairwise based on the proximity of the first and second components. This is because TWICE can bind two immune checkpoints after domain pairing. This would allow researchers to block the inactivation of IFN-γ receptors and result in robust immune cell activation.

[0515] Example 2. TWICE stimulates two pathways in immune cells Two pathways in immune cells (immune cell binding domain and complementary binding domain are paired) TWICEs that stimulate the IL-1 receptor (when activated) to mediate robust anticancer effects may be developed. For example, The cancer may be EGFR-expressing breast cancer.

[0516] In any of these examples, the second targeting moiety targets tumor microenvironment cells. In this example, the second targeting moiety can be selected to target a tumor-associated macrophage. Thus, the first component may be selected to target a transgenic phage (TAM). ) the first targeting moiety is capable of binding to an antigen expressed by a tumor, while (the second component The second targeting moiety (in the first group) can bind to an antigen expressed by the TAM. When targeting breast cancer expressing IL-1, the first targeting moiety can be cetuximab, and the second The second targeting moiety can be MAC-1 / CD11b.

[0517] FIG. 2 shows a first component targeted to cancer and a second component targeted to non-cancer cells (in this example, TAM This type of TWICE also targets TAMs. Domain pairing can only occur when the immune cell binding domain is in close proximity to the Pairing and complementary binding domain pairing are two pathways in immune effector cells. can be stimulated.

[0518] For example, one skilled in the art may choose to activate both the CD3e and CD137 pathways. To achieve this, the first component may be composed of the VH and VH sequences of SP34 (an anti-CD3 antibody). It contains the VL of utomirumab (anti-CD137 antibody) and the second component is SP34 (anti-CD3 A TWICE was created that contains the VL of tRNA (antibody) and the VH of utomirumab (anti-CD137 antibody). We can bring it out.

[0519] By creating TWICE in this way, TAMs in the vicinity, e.g., tumors, Both the first and second components of TWICE can be targeted to cancer cells in the stroma. Thus, the VH and VL of SP34 and the VH and VL of utomilumab are The pairing could be based on the proximity of the first and second components. This would allow for two positive stimuli to be provided to T cells after main pairing.

[0520] In a manner similar to that described in this Example or Example 1, an inhibitory checkpoint molecule (e.g., by paired immune cell binding domains) and T that both provides a positive stimulus (e.g., by pairing complementary binding domains) and These types of constructs could also generate robust anti-cancer immune responses. It can be activated.

[0521] Example 3. TWICE activates two different immune cells at the cancer site TWICE may also combine activities on two different immune effector cells For example, generating TWICE that mediates T cell activation and macrophage activation. is possible.

[0522] A targeting moiety against an antigen expressed by a cancer cell was prepared using a method as outlined in Example 1. In this particular example, both targeting moieties may be selected to target breast cancer cells. The two targeting moieties can bind to HER2 on the surface of the cells. R2. Thus, the ribozyme A receptor may bind to a different epitope on R2. Thus, the paired immune cell-binding domain of TWICE binds the first effector cell ( In the case of the first antibody, the complementary binding domain can bind to a second antibody (T cell), and the paired complementary binding domain can bind to a second antibody (T cell). The antibody may be one that binds to an effector cell (in this case a macrophage).

[0523] The present invention relates to a method for activating both the CD3 pathway of T cells and the CD40 pathway of macrophages. To do this, the first component may be SP34 (anti-CD3 antibody ) and VL of CP-870,893 (anti-CD40 antibody), and the second component The VL of SP34 (anti-CD3 antibody) and the VH of CP-870,893 (anti-CD40 antibody) You could produce TWICE that contains:

[0524] By creating TWICE in this way, the first and second components can bind to cancer cells. In cancer cells, the VH and VL of SP34 and CP-87 0,893 VH and VL can be paired based on the proximity of the first and second components. This suggests that TWICE mediates the expression of T cells and macrophages after domain pairing at the site of cancer. This would allow the phage to stimulate both.

[0525] In a similar way, researchers are trying to activate a combination of T cells and NK cells at the site of cancer. We could design TWICE to be like this.

[0526] Example 4. TWICE regulates immune cells and cancer cells TWICE combines both immune cell activity and direct activity in cancer cells. For example, immune checkpoint inhibitors expressed by cancer cells that mediate T cell activation may be involved. It is possible to create TWICE by blocking the intracellular molecule.

[0527] Two antibodies against a single antigen expressed by cancer cells, such as HER2 on breast cancer cells. The targeting moiety could be selected as outlined in Example 3. As shown in Figure 4, the paired immune cell-binding domain of TWICE binds to effector cells ( In this case, the complementary binding domains can bind to the cancer cells. The antibody may be one that binds to the cytoplasm.

[0528] One skilled in the art may choose to activate CD3 in T cells and block PD-L1 in cancer cells. To do this, the first component may be a V-type mAb of SP34 (anti-CD3 antibody). The second component is SP34 (anti-C TWICE containing the VL of D3 antibody and the VH of avelumab (anti-PD-L1 antibody) Let's create it.

[0529] By creating TWICE in this way, both components are targeted to cancer cells. In cancer cells, the VH and VL of SP34 and the VH of avelumab and VL could be paired based on the proximity of the first and second components. TWICE stimulates T cells in the tumor microenvironment after domain pairing and is expressed by cancer cells This would allow researchers to block expressed PD-L1-mediated immune suppression.

[0530] In a similar manner, we identified TWICE that binds to RANK or cell death-inducing antigens on cancer cells. It could be designed.

[0531] For example, we have demonstrated that CD3 activation on T cells directly induces cancer cell death. To do this, the first component may be SP34 (anti-CD3 antibody) The VH of the IL-16 antibody and the VL of mapatumumab (anti-TRAIL-R1 / DR4 cell death receptor antibody) were The second component contains the VL of SP34 (anti-CD3 antibody) and mapatumumab (anti-TRAI We were able to create a TWICE that contains the VH of the L-R1 / DR4 cell death receptor antibody. This suggests that TWICE stimulates T cells after domain pairing at the cancer site, This would make it possible to directly induce cancer cell death.

[0532] Thus, TWICE mediates direct anticancer effects as well as induces anticancer immune responses. It may also be stimulating.

[0533] Example 5. TWICE containing a dimerization domain In all of the constructs described in Examples 1 to 4, TWICE was dimerized as shown in FIG. One dimerization domain of the pair may also be linked to the immune cell binding domain. At the same time, the other dimerization domain of the pair can bind to the complementary binding domain. For example, each component may have a pair of knob-into-hole dimerization sequences. One or both of the dimerization domains may be linked by a cleavable linker (dimerization domain linker). For example, a linker containing an ADAM28 cleavage site (SEQ ID NO: 1) may be used. It may be possible to couple them via

[0534] In this way, the knob-into-hole dimerization domains of each component can be used to catalyze the dimerization of individual components. The immune cell binding domain of the antibody is forced to pair with the complementary binding domain. The cleavable linker connecting the merization domains allows the targeting moiety to target tumor microorganisms. After being directed to the environment, it could be cleaved to release the dimerization domain. Thus, the immune cell-binding domain and / or the complementary binding domain act as a signaling domain in the tumor microenvironment. and will be available for pairing with their corresponding domains in the other component.

[0535] Thus, dimerization domains can be used to target the target molecule outside the desired site of action in the tumor microenvironment. In this study, the immune cell binding domain of each component is forced to bind to the complementary binding domain, and the immune cell binding domain is forced to bind to the tumor microbe. This could prevent domain pairing that might occur between components outside of a small environment. In addition, cleavage of the dimerization domain linker by proteases in the tumor microenvironment may promote tumor Only in this case could domain swapping and acting take place.

[0536] Either both components contain a dimerization domain or only one component contains a dimerization domain. It would be possible to design TWICE that includes

[0537] Example 6. TWICE containing supplementary functional domains Other TWICEs have complement activity when directed to cancer cells, as shown in Figure 5. In this embodiment, the first component may comprise an attenuated IFN-alpha serotype. Itokine (Pogue et al., PLoS ONE 11(9):e01624 72(2016)).

[0538] Both the first and second components may comprise an anti-EPCAM scFv. is a 25-mer linker having an MMP2 cleavage site (AIPVSLR; SEQ ID NO: 46). Anti-CD3 tethered to an inactive binding partner VL domain derived from gantenerumab by E VH domains, such as those of SP34 (i.e., immune cell binding domains). The second component is a 25 meristematic protein with an MMP2 cleavage site (AIPVSLR; SEQ ID NO: 46). Inactive binding partner derived from cloned alpha-MUC1-1 antibody by r linker Anti-CD3E VL domains linked to VH domains, such as those of SP34 (i.e. immune cell binding domain).

[0539] In this manner, the immune cell binding domains of the first and second components bind to tumor-associated proteases. The first and second components are EPCA and are masked unless cleaved by the enzyme MMP2. When directed against M-positive tumors, cleavage by MMP2 occurs at the inactive binding domain (i.e., inactive In this way, the VH and VL of SP34 can mediate the release of the binding partner. In addition, the EPCAM antibody could activate T cells at the cancer site. By targeting weakened IFN-alpha to cancer cells, it has a direct anticancer effect. This type of TWICE may be able to suppress the anti-cancer effect by weakening cytokines. In addition to mediating these effects, it could also stimulate anti-cancer immune responses.

[0540] Similarly, TWICE recruits IL-15 or attenuated IL-2 to regulate T cell activation It may be possible.

[0541] In a similar manner, other attenuated cytokines can be incorporated as complementary functional domains in the first component. One could design a TWICE that contains the first and second components for additive effect. It may also be possible to design TWICE that contains complementary functional domains in both the .

[0542] Example 7. TWIs containing different dimerization domain linkers and different dimerization domains CE To construct an example of a TWICE molecule, we used the well-established anti-EpCA The EpCAM antibody solitomab (Brischwein K, et al. Mol Immunol. 43(8):1129-43(2006)) was selected, and this The antibody is composed of a heavy chain consisting of a heavy chain variable and a CH1 domain, and a light chain variable and a kappa constant domain. The antibody was used in the form of an antigen-binding fragment (Fab) containing two chains, one of which was a light chain consisting of

[0543] For the initial testing of these TWICE molecules, the VH of the anti-CD3 epsilon antibody SP34 was was fused to the C-terminus of the heavy chain of solitomab Fab, and a 15 amino acid long flexible linker was used. Using the anti-PDL1 antibody atezolizumab, the VL is fused to the C-terminus of the light chain of solitomab Fab. He made him do so.

[0544] As shown in Figure 6, the TWICE molecule ties together mispaired variable domains. Unless TWICE is proteolytically activated in the tumor microenvironment, The dimerization domain may contain a dimerization domain that prevents main exchange. to enhance efficient formation of heterodimers between the VH and VL domains, and To avoid the formation of homodimers, some heterodimers can be constructed. The heterodimerization domains have been described in the literature. To test whether the domain is suitable, several heterodimerization domains were used to generate the We constructed and expressed TWICE.

[0545] First, we synthesized TWI using a coiled-coil heterodimerization domain as shown in Figure 7A. CE was constructed. Coiled coils are also called leucine zippers. The sequences of the coiled-coil domains engineered as acidic and basic coils forming No. 20160002356A1 and US8877893B2. The heavy chain of the TWICE molecule is fused to a basic coil (SEQ ID NO: 201) and the light chain is fused to an acidic coil (SEQ ID NO: 202). The heavy chain was fused to the acidic coil (SEQ ID NO: 202). It will be appreciated that the strand may also be fused to a basic coil.

[0546] Table 12 shows the dimerization domain linkers contained in the coiled-coil TWICE constructs. These dimerization domain linkers are cleaved by proteases. The protease cleavage may result in the dimerization domain being split into two parts. This results in the separation of the two from the rest of the child by domain swapping. TWICE to allow for complementation of immune cell binding VH or VL domains. The examples given include some examples of dimerization domain linkers. In some cases, the linker is activated by matrix metalloproteinases in the tumor microenvironment. In another example, the dimerization domain linker contains a sequence that can be cleaved. Contains a protease site that can be cleaved by factor Xa (FXa), which It can be used to artificially activate the TWICE molecule in vitro. In this example, the factor Xa cleavage site is chosen because factor Xa is convenient for use in a laboratory environment. was employed as a surrogate for the protease cleavage site. In some of the examples listed in Table 12, The heavy and light chains of one TWICE molecule have the same promoter in their dimerization domain linker. In others, the heavy and light chains of one TWICE molecule contain a protease cleavage sequence. contain different protease cleavage sequences within their dimerization domain linkers.

[0547] In some of the examples listed in Table 12, TWICE acts as a dimerization domain to form heterodimers. In this case, the lysine residues disclosed in WO2017106462A1 are included. A layout strategy was used to construct TWICE with hetero-Fc dimerization domains. Table 1 2 is the dimer used for the TWICE molecules TWICE191 and TWICE192. TWICE191 has a heavy chain of SEQ ID NO: 171 and a covalent domain linker of SEQ ID NO: 17 TWICE192 comprises a heavy chain of SEQ ID NO: 173 and a light chain of SEQ ID NO: 174. These TWICEs contain the mutant S to promote the formation of hetero-Fc between the heavy and light chains. 364K / K409L in the heavy chain Fc and K370S / F405K in the light chain Fc Designing TWICE with a hetero-Fc dimerization domain is a molecular It binds to the fetal Fc receptor, FcRn, which mediates the recycling of FcR and extends its serum half-life. These TWICE examples are non-glycosylated TWICEs with weakened effector functions. It further contains a N297Q mutation in the Fc resulting in a lysed molecule.

[0548] In another example, the Fab constant domains (CH1 and CL) are used as dimerization domains. The TWICE molecule was constructed using the Fab fragment as a cancer targeting moiety. To allow efficient assembly of the heavy and light chains of the molecule, the dimerization domains CH1 and CL are CrossFab was constructed by linking VH to CL and VL to form a crossFab (Figure 7A). Bispecific antibodies can be produced by cross-linking the constant domains in one of the Fab fragments to link the Fab fragment to CH1. This strategy allows for efficient assembly of the two light chains of the fusion construct (Schaefer Weimann, t a., Proc Natl Acad Sci USA.108(27):1118 7-92(2011)). TWICE construct containing CrossFab dimerization domain In the present study (TWICE200 and TWICE204, see Table 13), immune cell engagers The VH of the CL domain is connected to the VH of the CL domain by a dimerization domain linker sequence, and the complementary endonucleases are The VL of Gager is connected to the CH1 domain by a dimerization domain linker sequence All dimerization domain linkers contain the factor Xa protease cleavage sequence (Ile-Glu- The complete sequence of TWICE200 contains the heavy and and a light chain of SEQ ID NO: 178, and construct TWICE204 contains a heavy chain of SEQ ID NO: 179 and a light chain of SEQ ID NO: 179. Contains the light chain of sequence number 180.

[0549] In another example, TWICE was synthesized using the IgE CH2 domain as a dimerization domain. IgE CH2 normally functions as a hinge domain in IgE, but domains to construct Fab-like molecules with similar binding properties by domain replacement (Cooke et al., mAbs. 10(8):1248-1 259 (2018)). In TWICE189 and TWICE190, the dimers shown in Table 12 were The IgE CH2 domain is linked to the immune cell engager using a somatic domain linker sequence. The dimerization domain linker was a factor Xa cleavage sequence ( It contains the amino acid sequence Ile-Glu-Gly-Arg.

[0550] [Table 12]

[0551] [Table 13]

[0552] The TWICE proteins listed in Table 13 were co-transfected with the respective heavy and light chains. The expression was performed in transient HEK293 cultures (30-50 ml) in shake flasks by The expressed protein was purified from the supernatant using an IMAC column (Histrap Excel, G E) by FPLC using ELISA or, in the case of Fc-containing TWICE proteins, by protease inhibition. The purified product was purified using a TEIN A column (MabSelect PrismA, GE). The proteins were analyzed by SDS-PAGE and all linkers and dimers tested were A band was observed at the predicted molecular weight for the transcription domain (Fig. 7B). The TWICE with coiled-coil or hetero-Fc dimerization domains is the best. was also expensive.

[0553] TWICE was further tested by cleavage with recombinant FXa protease. In the reaction system, approximately 1 μg of purified TWICE and 0.1 μg of FXa (New En gland Biolabs #P8010) in 100 mM NaCl and 2 mM C aCl2 and 20 mM Tris-HCl (pH 8.0) in a buffer containing ambient The plates were incubated at room temperature for 24 h in 4× LDS sample buffer (Thermo Fisher Scientific). The reaction was stopped by adding 5 μl of NP0007 and heating to 95°C for 2 min. The samples were then separated by SDS PAGE and the cleavage products were analyzed (Figure 1). 7C).

[0554] Both dimerization domain linkers contain the FXa cleavage sequence in the heavy and light chains. Samples TWICE193, TWICE191, and TWICE192 (Table 13) have FX a (Figure 7C). FXa cleavage in the heavy chain dimerization domain linker TWICE11 with an MMP2 cleavage site in the light chain dimerization domain linker 7 was only partially cleaved (Figure 7B). Construct TWICE200, which has an FXa cleavage site contained within TWICE 204, TWICE189, and TWICE190 showed no FXa protein expression under these conditions. This result suggests that the dimerization domain arrangement (CH1 / CL , or IgE CH2), or constructs TWICE200, TWICE204, TWI The linker lengths of 5 to 8 residues in CE189 and TWICE190 (Table 12) were These results suggest that these particular constructs are not favorable for protease cleavage. While designing TWICE117, TWICE193, TWICE192 and TWI The dimerization domains contained in CE193, coiled-coil and hetero-Fc, are TW TWICE molecules containing these dimerization domains are suitable for constructing ICE. and could be activated by cleavage with a recombinant protease.

[0555] Example 8. TWICE with different binding moieties: Fv vs. Fab Hetero-Fc dimerization domain with CD3 antibody SP34 and CTLA4 antibody ipilimumab In some cases, the anti-EpCAM antibody solitomab was used. was used as Fab (TWICE277 and TWICE278), and in other examples this antibody A disulfide bond is formed between residue 44 of VH and residue 100 of VL (represented by Kabat numbering). Disulfide-stabilized Fv (dsFv, TWICE332 and TWI TWICE molecules were expressed as described above and bound to Protein A. The antibody was purified by affinity chromatography and its binding ability to EpCAM was assayed by ELISA. The ELISA plates were then loaded with recombinant soluble EpCA (produced in-house). Plates were coated with M-His6 protein at 5 μg / ml overnight at 4°C. The cells were blocked with 1% BSA in PBS. Serial dilutions of TWICE bound to EpCAM. The bound TWICE was then incubated with a mouse anti-human Fc secondary antibody (JDC-10, Abcam, The results (Figure 8A-B) show that Fab or dsFv targeting The TWICE molecules with the tRNA-binding portion bind to the antigen with similar binding affinity and the KD is The range for TWICE was 1.04–1.65 nM, whereas the range for Fab TWICE was 1.21 The results showed that the IL-10 binding sites were in the range of ~1.22 nM. Thus, the IL-10 binding sites were targeted to cells in the tumor microenvironment. To achieve this, TWICE can be combined with a different targeting moiety, e.g., Fab or dsFv. It can be constructed.

[0556] Example 9. Testing of CD3 / CTLA-4 TWICE in a bridging ELISA Domain swapping and functional paratopes (i.e. paired VH / V To test the production of the L domain, the target antigen and the immune cell-engaging molecule are simultaneously bound to each other. A bridging ELISA assay was developed to measure the ability of the TWICE pair to bind to the ribosome. The principle of this bridge ELISA is as follows. In brief, the ELISA plate is coated with the target antigen ( The wells were then coated overnight with recombinant soluble EpCAM (black hexagons) at a concentration of 5 μg / ml. The TWICE molecule can be activated by cleavage with FXa as described above, followed by stepwise dilution. TWICE can be added to the plate in the same manner as Fab (four grey ovals) in Figure 9A. The target antigen can be bound by its targeting moiety, which is depicted as Binding to the target antigen (EpCAM) on the chromosome brings TWICE into close proximity, which is essential for immune cell binding. The domain and the complementary binding domain undergo domain exchange to form a complete immune cell-binding paratope ( Functional complementation allows immune cells to form The antigen bound by the binding domain, e.g., CD3 (striped triangle in FIG. 9A), The antigen is an Fc fusion protein (mouse IgG2a Fc) and can therefore be detected with an anti-mouse Fc secondary antibody. This detection step with a secondary antibody has been omitted for clarity in the schematic diagram of Figure 9A. There are.

[0557] Figure 9B and Figure 9C show cross-linking E to measure functional complementation of TWICE pairs by domain swapping. Examples of LISA are shown below. In these examples, TWICE 277 and 278 (listed in Table 13) These include the immune cell-binding antibody SP34, which binds to CD3 epsilon, and It contains the complementary antibody ipilimumab, which binds to CTLA-4. The VH and VL of the CTLA-4 antibody are contained in TWICE277, and the VL and VL of the anti-CD3 antibody are The VH of the TWICE molecule is the VH of the anti-CTLA-4 antibody. Examples include heterodimerization domains and FXa in both dimerization domain linkers. It further contains a cleavage site. Figure 9B measures the binding ability of TWICE to CD3. Results from the CD3 cross-linking ELISA are shown in Figure 9C. The results of a CTLA-4 bridging ELISA measuring binding capacity are shown. WICE (277 or 278 alone) was activated by cleavage with FXa. Even when T When WICE277 and 278 were used in combination, they did not activate the cells by cleavage with FXa. In this case, TWICE did not bind to either CD3 or CTLA-4. ICE277 and 278 were used in combination and cleaved with FXa to remove the dimerization domain Only when CTLA-4 was activated did they respond in a dose-dependent manner to CD3 or CTLA-4 binding. These results indicate that both TWICEs are targets. Upon antigen binding, TWICE molecules undergo domain exchange to form functional immune cell-binding domains. Furthermore, the dimerization domains of these TWICEs can generate As intended, TWI does not bind to the dimerization domain unless it is removed by protease cleavage. It acts as a locking mechanism to prevent CEs from swapping domains.

[0558] 10A-10B show the CD3 / CTLA-4 TWIs tested in bridging ELISA experiments. Another example of CE is shown below. TWICE281 and 282 used in this experiment are the same as those of the above TWI Same EpCAM tropism as CE277 and TWICE278, and CD3 and CTLA- 4 antibody, but differs in that it contains a coiled-coil dimerization domain. In addition, TWICE281 and 282 contain an FXa cleavage site in the heavy chain and an MMP2 cleavage site in the light chain. It contains a cleavage sequence, and therefore the molecule is activated by cleavage by FXa or MMP2. As in the previous experiment, TWICE277 and 278 were cleaved by FXa. Only when activated with FXa did TWI show a dose-response binding curve. CE did not bind to either CD3 or CTLA-4. Thus, heterologous Fc and coil Both coil dimerization domains are suitable for the design of TWICE. A hetero-Fc may be preferred if extended half-life is desired.

[0559] Example 10. CD3 / CD28 TWICE 11A-11B show the CD3 antibody SP34 and co-immunoglobulin (C-Ig) binding assays tested in bridging ELISA experiments. An example of TWICE constructed from the potent anti-CD28 antibody celalizumab is shown (T These examples of TWICE again have both heavy and light chain linkers. The antibody was designed with a hetero-Fc dimerization domain and an FXa cleavage site. Similarly, TWICE394 and TWICE395 are activated by cleavage when used in combination. Only when the antibody was activated, it showed binding to the immune cell antigens CD3 and CD28 (Figure 1 1A and 11B). Individual TWICEs did not show binding to either immune cell antigen. As evidenced by the fact that uncleaved TWICE did not exhibit any cross-linking activity, Again, cleavage was required for domain swapping and binding (Fig. 11A and Fig. 11 (Gray square in B).

[0560] Example 11. CD28 / 4-1BB TWICE 12A-12B show a marker that binds to the costimulatory molecules CD28 and CD137 (4-1BB). An example of TWICE constructed using the antibodies ceralizumab and urelumab is shown. TWICE400 and 401 (shown in Table 13) are potential candidates for ceralizumab and urelumab. The FX domain was constructed using a heterodimerization domain and a heterodimerization domain in both linkers. These molecules were tested in a bridging ELISA format and showed that the TW ICE inhibited CD2 when the construct was precleaved with FXa and both TWICEs were present. 8 (FIG. 12A) and 4-1BB (FIG. 12B). E alone also did not bind to either CD28 or 4-1BB.

[0561] Example 12. CTLA-4 / PD-L1 TWICE Figure 13 shows the binding of the checkpoint molecules CTLA-4 and PD-L1, respectively. An example of TWICE constructed using the antibodies ipilimumab and atezolizumab is shown. As shown in Table 13, TWICE283 has the VH of ipilimumab and the VL of atezolizumab. The complementary TWICE284 contains the VH of atezolizumab and the VL of ipilimumab. These TWICEs contain a coiled-coil dimerization domain and a FXa domain in the heavy chain. These TWICEs were designed to have a cleavage site for EpCAM / PD-L1 crosslinking. TWICE284 was tested by ELISA and found to be a potent anti-atezolizumab inhibitor. Despite only containing VH and lacking the cognate atezolizumab VL, PD- The results showed that the IL-1 binding domain was involved in the mediation of antigen binding. This suggests that the VH of tedolizumab is sufficient.

[0562] This view is based on the idea that the VL of an antibody does not make many contacts with the antigen and is therefore potentially dispensable in antigen binding. or may be replaceable, as shown in the crystal structure of atezolizumab in complex with PD-L1 (L From ee HT et al. Sci Rep 7:5532-5532 (2017) This is consistent with the current evidence. When used in combination with ipilimumab, the paratope (i.e., VH The construction of TWICE due to the fact that the TWICE / VL pair retains its binding properties when split. This is an example of an antibody that is not suitable for

[0563] However, as shown in Figures 9A to 12B, ipilimumab, SP34, and ceralizumab and urelumab are exemplary antibodies that do not exhibit binding when the paratope is disrupted. Such antibodies are therefore suitable for the construction of TWICE.

[0564] Example 13. Immune cell activation by TWICE T cell targeting by the TWICE construct was examined in an in vitro T cell activation assay. Briefly, colon cancer cells (HCT-15) were cultured in a 96-well plate at 5,000 Peripheral blood mononuclear cells (PBMCs) were plated at a density of 1000 cells / well and allowed to adhere overnight. Cells were treated with serial dilutions of TWICE molecules, added at an effector-to-target ratio of 10:1. In some experiments, TWICE molecules were incubated with FXa protease prior to addition to cells. It was preactivated by cleavage with (NEB#P8010L).

[0565] To quantify T cell activation, an IFN-gamma ELISA kit (Invitrogen) was used. n 88-7316-88) was used to measure the concentration of TWICE in the medium 24 hours after the addition of TWICE. The secreted interferon gamma was detected. Lactate dehydrogenase (LDH) was detected in the lysed cancer cells. We used a cytotoxicity assay (CytoTox96, Promega) to quantitatively measure Target cell killing was assessed 48 hours after the start of treatment. The dose-response curves shown in Figure 6 are expressed as % specific lysis, which was measured using lysis buffer. It corresponds to the percentage of LDH release compared to cells lysed with 100 mM sodium phosphate ... minus round LDH release.

[0566] 14A-14B show TWICE 277 and 278, which were previously tested in a bridging ELISA. The results of the cell killing assay using TWICE281 and 282 are shown. These two antibodies were anti-CD3 epsilon-engaging antibodies SP34 and CTLA-4 TWICE277 and 278 contain the FXa cleavage sequence Ile- The linker GGGIEGRGGG (SEQ ID NO: 206) were constructed with a heterodimerization domain linked to VH or VL. In contrast, TWICE281 and 282 contain a coiled-coil dimerization domain. The linkers in the heavy and light chains are not identical (FIG. 14A). The cleavage site is in the heavy chain linker GGGGSIEGRGGGGS (SEQ ID NO: 203). The MP2 cleavage site is contained in the light chain linker GGPLGVRGKGGGS (SEQ ID NO: 204). The LDH release data from this experiment (Figure 14B) demonstrates that TWICE is a Both TWICE pairs were P when preactivated with FXa prior to addition to cells. The results showed that BMC caused strong killing of cancer cells. 78 did not elicit this killing response without prior cleavage, suggesting that TWIC These results support the requirement of cleavage for E activation and domain swapping, and we used these molecules to This is consistent with the data obtained in the bridging ELISA experiments (Figures 9B-9C). However, in the bridging ELISA (Fig. 10A-10B), activation by FXa was also required. TWICE281 and 282 are the ones that were created when TWICE was not activated beforehand. Even the combination of 100 and 150 mg of 100% naphthalene rapidly induced killing of HCT15 cells by PBMC in a dose-dependent manner.

[0567] These results suggest that the MMP2 site in the light chain linker can be cleaved by cancer cells. Overall, this experiment shows that (1) (2) TWICE can efficiently engage with immune cells and kill cancer cells; (3) TWICE activation requires proteolytic cleavage and is upregulated by cancer cells. This confirms that the protease produced by the enzyme can activate the TWICE molecule. Ta.

[0568] 15A-15D also show anti-CD3 epsilon-engaging antibody SP34 and anti-CTLA-4 antibody TWICE285 and TWICE286, which contain ipilimumab, were used. Cleavage assay (Figure 15A), T cell activation assay (Figure 15B) and killing assay (Figure 1 5C to 15D) are shown. These examples of TWICE show that Within the dimerization domain linker GGGGSIEGRGGSGGGS (SEQ ID NO: 205) The FXa cleavage site is designed to be These examples of TWICE also contain a coiled-coil dimerization domain. Contains.

[0569] When TWICE285 was cleaved with FXa, the molecule gave the expected fragments on SDS-PAGE. We have previously generated the results (Figure 15A) and demonstrated that TWICE can be preactivated with FXa. The cytokine release and killing data shown in Figures 15B and 15C are WICE285 and TWICE286 are cleaved by FXa protease to form TWICE When E was activated, it engaged and redirected PBMCs to HCT-15 cells. Neither TWICE285 nor TWICE286 alone can inhibit T cells. It did not cause the release of interferon gamma or the killing of cancer cells.

[0570] However, these TWICEs were combined without prior cleavage in LDH release experiments. When tested, the molecule caused cancer cell lysis regardless of prior activation with FXa ( (Figure 15D). This result was consistent with the results of cross-linking ELISA and T cell activation assays. This is in contrast to the TWICE example above, where a cut was required. The data demonstrate that the design of the dimerization domain and linker length is relevant. The longer linker GGGGSIE unique to TWICE285 and TWICE286 GRGGSGGGS (SEQ ID NO: 205) represents the coiled carp gene present in these constructs. When used in combination with a cyclic dimerization domain, it may not prevent domain swapping in the absence of cleavage. There is a gender.

[0571] Example 14. Costimulatory TWICE Next, TWICE3, which binds to CD3 and CD28 as shown in Table 13 94 and TWICE395 were tested in a T cell directed redirection assay (FIG. 16). As shown, these TWICEs were composed of the anti-CD28 antibody ceralizumab and the anti-CD3 epsilon antibody These molecules are therefore constructed from the structure SP34 shown in Figures 11A-11B. It can bind to CD3 and CD28 in a manner that induces T cell activation. These TWICEs were tested in a T cell killing assay. TWICE394 and 395 inhibited the expression of FXa in HCT-15 cancer cells after preactivation with FXa. It caused robust killing but was not active without prior cleavage.

[0572] To test the stimulatory signal induced by the engagement of CD28 and CD3 by TWICE, We developed a protocol to exhaust T cells before testing for WICE activity. For the purpose of incubation and exhaustion, prostate cancer cells (PC-3) were plated in 6-well plates at 150,000 cells / well. Peripheral blood mononuclear cells (PBMCs) were seeded at a density of 10 cells / well and allowed to adhere overnight. SEQ ID NO: 168 of US10035856B2 and Cells were treated with a 169-based two-component T cell engager at a concentration of 10 nM. The two-component system is similar to CD3 bispecific antibodies in that T cells are engaged to kill cancer cells. However, it has a similar effect to conventional bispecific antibodies in terms of preactivation. This has the advantage that the molecule cannot bind to CD3 in solution, and therefore Therefore, they can be easily removed by washing the PBMCs. PBMCs were analyzed by flow cytometry 48 hours after activation to determine whether the treatment had activated or inhibited PBMCs. and CD3+ cells, causing robust upregulation of exhaustion markers ( Figure 17A) revealed preactivation and exhaustion. Such preactivated immune cells were These may better reflect immune cells in the tumor microenvironment compared to stimulated PBMCs. It is known that tumor-infiltrating lymphocytes are exhausted and show increased expression of exhaustion markers. (Baitsch L et al. Trends Immunol .33(7):364-72(2012)).

[0573] The pre-activated PBMCs were then redirected to the above colon cancer cells (HCT-15). The assay was used to test the possibility that the TWICE molecule induces T cell activation, but In this assay, the effector to target ratio was changed to 1:4. The results of the activation assay are shown. TWICE394 and TWICE394 bind to CD3 and CD28. ICE395 induces robust activation of exhausted T cells as measured by IFN-gamma secretion TWICE277 and TWICE2 bind to CD3 and CTLA-4. Although the level of 78 is lower than that of TWICE394 and TWICE395, The CD3 antibody VH and VL domains were converted to non-functional VH and VL domains. A pair of control TWICEs containing the L domain (SEQ ID NOs: 199 and 200) were also identified in this article. The controls, TWICE431 and TWICE432, were used to treat exhausted T cells. In this redirection experiment, low levels of interferon gamma were produced. Overall, these results demonstrate how trispecific antibodies can activate CD3 and CD28 together. The results showed that it produced superior T cell activation compared to either stimulation alone and that it was more potent. This is consistent with recent publications showing that tumor-specific immune responses can be developed into antitumor immune responses (Wu, L., Seung, E., Xu, L. et al. es enhance the therapeutic efficacy of t umor-directed T cells through T cell rec eptor co-stimulation.Nat Cancer (November 2019) (Published online on the 18th) doi:10.1038 / s43018-019-0004 Consistent with this finding, our data demonstrate that TWICE can potently stimulate exhausted T cells. and the additive effects of costimulation or checkpoint blockade on CD3 engagement alone. However, the results suggest that the three published In contrast to bispecific antibodies, TWICE mediates these costimulatory signals in a manner that can activate them. can be provided.

[0574] Example 15. TWICE Engaging NK Cells via CD16A In another example, we used an antibody against CD16A to target TWICE to natural killer (NK The antibodies used to construct these TWICE molecules were designed to engage cells. The CD16A antibody was shown to inhibit CD30 and CD1 The molecule AFM13, a bispecific antibody targeting 6A, has been evaluated. To evaluate this antibody in We created the TWICE molecules TWICE440 and TWICE441, which , a hetero-Fc dimerization domain, and a FXa cleavage site in the dimerization domain linker. It was something that could be done.

[0575] TWICE molecules were first analyzed by CD16A cross-linking ELISA, similar to the cross-linking ELISA experiment described above. TWICE440 and 441 were tested. Both TWICEs were present and the FXa protease was Expected binding to CD16A only when preactivated by ribosomal enzyme As seen for other TWICE molecules, the prior Cleavage is required, and uncleaved TWICE binds to CD16A in this cross-linking ELISA assay. Furthermore, individual TWICE molecules did not exhibit binding to FXa. Even when precleaved, AFM1 did not show binding to CD16A. The anti-CD16A antibody derived from 3 has split paratope halves (i.e., VH and VL) does not bind to antigens during separation in TWICE, so it is not used in TWICE. It is suitable for

[0576] Subsequently, TWIC was identified in a targeting assay using HCT15 colon cancer cells and NK cells. E440 and 441 were tested in the targeting assay performed using PBMCs as described above. Cancer cells were seeded at 5,000 cells / well in 96-well plates as described above. Next, purified NK cells (Stem Cell Technology) were seeded on the cells and allowed to adhere overnight. logies, Catalog No. 70036) to cancer cells at an effector-to-target ratio of 20:1 TWICE was added to the wells at various concentrations. After 24 hours, the cytotoxicity assay was performed to quantitatively measure LDH from lysed cancer cells. The target cells were then transfected with a cytotoxin (CytoTox96, Promega) to measure LDH release. The results of this NK cell directed targeting assay are shown in Figure 18B. A control bispecific antibody targeting EpCAM and CD16a (CTRL406 ) induced the release of LDH from cancer cells, indicating their killing by NK cells.

[0577] A control bispecific antibody targeting EpCAM and CD3 also likely inhibited some NKT The extent is smaller than that of the CD16a / EpCAM bispecific antibody due to the presence of cells. Although the expression of TWICE440 and 441 was not significant, they did induce LDH release. This was observed with a CD16a / EpCAM bispecific antibody. The amount of killing was comparable to that of TWICE molecules that engage NK cells, and was inhibited by CD16a antibodies. It was shown that anti-CD16 antibody-mediated NK cell engagement can promote cancer cell killing. can be caused.

[0578] Equivalent The above specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. It is contemplated that the above description and examples detail certain specific embodiments and the present inventors However, the above description may not be sufficient to fully explain the best mode contemplated by the present invention. Although it may appear that the embodiments are illustrative, they may be practiced in many ways and are within the scope of the appended claims. It will be understood that the terms "common" and "non-common" should be interpreted according to the scope and any equivalents thereof.

[0579] As used herein, the term "about" means any number of substances, regardless of whether or not expressly indicated. The term "about" generally refers to numerical values ​​including, but not limited to, whole numbers, fractions, and percentages. , which would be recognized by one of ordinary skill in the art as being equivalent to the recited values ​​(e.g., having the same function or result). Refers to the range of values ​​considered (e.g., ±5-10% of the listed range). When a term such as "about" precedes a recited value or range, the term In some cases, the term "about" modifies any listed value or range. , may include numerical values ​​rounded to the nearest significant figure.

Claims

1. 1. A kit or composition for treating cancer in a patient, comprising: a. a first component comprising a targeted immune cell binding agent, said targeted immune cell binding agent comprising: i. a first targeting moiety that binds to a tumor antigen expressed by the cancer; ii. a first immune cell binding domain, either a VH domain or a VL domain, that is capable of immune cell binding activity when bound to a second immune cell binding domain that is not part of the first component; iii. a first inert binding partner of the first immune cell binding domain, which binds to the first immune cell binding domain such that the first immune cell binding domain does not bind to the second immune cell binding domain unless the inert binding partner is removed, the first inert binding partner being a VL domain when the first immune cell binding domain is a VH domain, and a VH domain when the first immune cell binding domain is a VL domain; iv. a protease cleavage site separating the first immune cell binding domain and the first inactive binding partner, (1) expressed by the cancer or tumor microenvironment cells; or (2) (a) is the same as or different from the first and / or second targeting moieties in the targeted immune cell binding agent and binds to a tumor antigen expressed by the cancer; or (b) is co-localized to the cancer by a targeting moiety that is an antibody or antigen-binding fragment thereof that binds to an antigen expressed by cells in the tumor microenvironment. the protease cleavage site capable of releasing the inactive binding partner from the immune cell binding domain in the presence of a protease which is v. A first complementary functional domain capable of immune cell binding. Including, the first component, and b. a second component comprising a targeted immune cell binding agent, said targeted immune cell binding agent comprising: i. a second targeting moiety; ii. a second immune cell binding domain, and iii. the second component optionally comprising a second complementary functional domain capable of immune cell binding; The kit or composition comprising:

2. 2. The kit or composition of claim 1, wherein the second component comprises a complementary functional domain, and optionally the complementary functional domain of the first and / or second component comprises a ligand or agonist of a receptor, and further optionally the complementary functional domain comprises a latent form of a member of the TGF-beta family or a cytokine.

3. 3. The kit or composition of claim 2, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or a member of the TNF superfamily.

4. 3. The kit or composition of claim 2, wherein the complementary functional domains of the first and / or second components comprise an attenuated cytokine, optionally wherein the attenuated cytokine is a variant of a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

5. 3. The kit or composition of claim 2, wherein the complementary functional domains of the first and / or second components comprise an agonist that binds to an IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily receptor, and optionally, the agonist is an antibody or antigen-binding fragment thereof.

6. 10. The kit or composition of claim 1, wherein the second targeting moiety binds to a tumor antigen expressed by the cancer.

7. the second component further comprises a second inert binding partner of the second immune cell binding domain, wherein the second inert binding partner binds to the second immune cell binding domain such that the second immune cell binding domain does not bind to the first immune cell binding domain unless the inert binding partner is removed, and wherein the inert binding partner is a VL domain when the second immune cell binding domain is a VH domain, and the inert binding partner is a VH domain when the second immune cell binding domain is a VL domain; and a protease cleavage site separates the second immune cell binding domain and the second inactive binding partner, the protease cleavage site comprising: a. Expressed by said cancer, or b. (a) is the same as or different from the first and / or second targeting moiety in the agent and binds to a tumor antigen expressed by the cancer, or (b) is co-localized to the cancer by a targeting moiety that is an antibody or antigen-binding fragment thereof that binds to an antigen expressed by cells in the tumor microenvironment.

14. The kit or composition of claim 13, wherein the inactive binding partner can be released from the immune cell binding domain in the presence of a protease which is

8. 2. The kit or composition of claim 1, wherein the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to a T cell, a macrophage, or a natural killer cell.

9. 9. The kit or composition of claim 8, wherein the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to a T cell, and optionally the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to CD3, a T cell receptor, programmed death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD28, CD137, OX40, CD27, GITR (TNFRSF18), TIGIT, or inducible T-cell costimulatory factor (ICOS).

10. The kit or composition described in claim 8, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to macrophages when bound to each other, and optionally the first immune cell binding domain and the second immune cell binding domain are capable of binding to CSF1R when bound to each other.

11. 9. The kit or composition of claim 8, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to a natural killer cell when bound to each other, and optionally the first immune cell binding domain and the second immune binding domain are capable of binding to CD16A when bound to each other.

12. wherein the first and / or second targeting moiety comprises an antibody or antigen-binding fragment thereof, and optionally the first and / or second targeting moiety is selected from the group consisting of alpha4 integrin, A33, ACVRL1 / ALK1, ADAM17, ALK, APRIL, BCMA, C242, CA125, cadherin-19, CAIX, CanAg, carbonic anhydrase IX, CCN1, CCR4, CD123, CD133, CD137 (4-1BB), CD138 / syndecan-1, CD19, CD2, CD20, CD22, CD30, CD33, CD37, CD38, CD4, CD40 , CD44, CD45, CD48, CD5, CD52, CD56, CD59, CD70, CD70b, CD71, CD74, CD79b, CD80, CD86, CD98, CEA, CEACAM, CEACAM1, CK8, c-Kit, CLDN1, CLDN18, CLDN1 8.2, CLDN6, c-met / HGFR, c-RET, Cripto, CTLA-4, CXCR4, DKK-1, DLL3, DLL4 , TRAIL-R2 / DR5, DRS, EGFL7, EGFR, EGFRvIII, endoglin, ENPP3, EpCAM, EphA2, Episialin, FAP, FGFR1, FGFR2, FGFR3, FGFR4, fibronectin extra domain B, FLT-3, flt4, folate receptor 1, GCC, GD2, GD3, glypican-3, glypican, GM3, GPNMB, GPR49, GRP78, Her2 / Neu, HER3 / ERBB3, HLA-DR, ICAM-1, IGF-1R, IGFR, IL-3Ra, integrin α5β1, integrin α6β4, integrin αV, integrin αVβ3, Lewis Y, Lewis y / b antigen, LFL2, LIV-1, Ly6E, MCP- 1, mesothelin, MMP-9, MUC1, MUC18, MUC5A, MUC5AC, myostatin, NaPi2b, neuropilin 1, NGcGM3, NRP1, P-cadherin, PCLA, PD-1, PDGFRa, PD-L1, PD-L2, phosphatidylserine, PIVKA-II, PLVAP, PRLR, progastrin, PSCA, PSMA, RANKL, RG1, Siglec-15, SLAMF6, SLAMF7, SLC44A4, STEAP-1, TACSTD-2, tenascin-C, TPBG, TRAIL-R1 / DR4, TROP-2,The kit or composition of claim 1, comprising an antibody or antigen-binding fragment thereof that binds to TWEAKR, TYRP1, VANGL2, VEGF, VEGF-C, VEGFR-2, or VEGF-R2.

13. The first and / or second targeting portion is an anti-α4 integrin antibody, an anti-CD137 antibody, an anti-CCR4 antibody, an anti-CD123 antibody, an anti-CD133 antibody, an anti-C D138 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD49d antibody, anti-CD52 antibody , anti-CD70 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD80 antibody, anti-CEA antibody, anti-cMet antibody, anti-Cripto antibody, anti-CTLA-4 antibody, anti-D LL3 antibody, anti-TRAIL-2 / DR5 antibody, anti-E-cadherin antibody, anti-endoglin antibody, anti-EpCAM antibody, anti-epidermal growth factor receptor antibody, anti-FGFR3 antibody , anti-fibronectin extra domain B antibody, anti-folate receptor 1 antibody, anti-glypican 3 antibody, anti-gp95 / 97 antibody, anti-Her2 antibody, anti-IGF-1R antibody, anti-IL-13R antibody, anti-IL-4 antibody, anti-IL-6 antibody, anti-MMP-9 antibody, anti-MUC1 antibody, anti-mucin core protein antibody, anti-NGcGM3 antibody, anti-P-cadherin antibody, anti-PD-L1 antibody, anti-p-glycoprotein antibody, anti-PSCA antibody, anti-PSMA antibody, anti-SLAMF7 antibody, anti-TRAIL-R1 / DR4 antibody, anti-transferrin antibody, anti-TROP-2 antibody, or anti-VEGF antibody, or an antigen-binding fragment thereof.

14. The first and / or second targeting moiety is selected from the group consisting of alemtuzumab, andecaliximab, atezolizumab, avelumab, BCD-100, bevacizumab, BGB-A317, blinatumomab, brentuximab, BU59, camrelizumab, carotuximab, catumaxomab, cemiplimab, cetuximab, daratumumab, depatuxizumab, dinutuximab, DS-8201, durvalumab, edrecolomab, elotuzumab, G544, gemtuzumab, glembatumumab, GP1.4, hp67.6, IBI308, ibritumomab, inotuzumab, ipilimumab, isatuximab, and L19IL 2, L19TNF, margetuximab, mirvetuximab, mogamuizumab, moxetumomab, natalizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumumab, oportuzumab, panitumumab, PDR001, pembrolizumab, pertuzumab, polatuzumab, racotumomab, ramucirumab, rituximab, rovalpituzumab, sacituzumab, SM3, TAK-164, tositumomab, trastuzumab, tremelimumab, ublituximab, urelumab, utomilumab, XMAB-5574, or zolbetuximab.

15. The kit or composition of claim 1, wherein the first and / or second targeting moieties comprise a DNA aptamer, an RNA aptamer, albumin, lipocalin, fibronectin, ankyrin, finomer, obody, DARPin, knotin, avimer, atrimer, anti-callin, affilin, affibody, bicyclic peptide, cys-knot, FN3 (adnectin, centryrins, pronectin or TN3), or a Kunitz-type domain.

16. The first and / or second targeting moiety: a. IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD-1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40 b. the full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folate, EGF, TGF, PD-1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40; or c. Truncated forms, analogs, variants or derivatives of IL-2, IL-4, IL-6, α-MSH, transferrin, folate, EGF, TGF, PD-1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40 2. The kit or composition of claim 1, comprising:

17. 2. The kit or composition of claim 1, wherein the first and / or second targeting moiety binds to IL-2 receptor, IL-4, IL-6, melanocyte-stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.

18. 2. The kit or composition of claim 1, wherein the second targeting moiety binds to an antigen expressed by a tumor microenvironment cell, optionally the tumor microenvironment cell is a fibroblast or a macrophage, and further optionally, the tumor microenvironment cell is a fibroblast and the antigen expressed by the fibroblast is a fibroblast activation protein.

19. 19. The kit or composition of claim 18, wherein the antigen expressed by fibroblasts is fibroblast activation protein, or the antigen expressed by macrophages is MAC-1 / CD11b or sideroflexin 3.

20. 10. Use of the kit or composition of claim 1 in the manufacture of a medicament for delivering a cytokine to an immune cell, wherein the first and / or second supplemental functional domains of the composition comprise IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or a member of the TNF superfamily.

21. Use of the kit or composition of claim 1 in the manufacture of a medicament for delivering a cytokine receptor agonist to an immune cell, wherein the first and / or second supplemental functional domains comprise a cytokine receptor agonist that binds to an IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ or TNF superfamily receptor, and optionally, the agonist is an antibody or an antigen-binding fragment thereof.

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