Dual immune cell adapter

Abstract

Double immune cell connector (TWICE) is a kit or composition for treating cancer comprising a first component and a second component. Each component comprises a targeting portion for binding to a tumor antigen expressed by the cancer or an antigen expressed by a non-cancerous cell in a tumor microenvironment. In some embodiments, each of the first component and the second component comprises an immune cell binding domain capable of having immune cell binding activity when combined with an immune cell binding domain in another component, and a complementary binding domain capable of binding to a complementary antigen when combined with a complementary binding domain in another component. In some embodiments, the first component and / or the second component comprises a complementary functional domain that is active when targeted to a cancer cell or its microenvironment. The TWICE complex of the present invention provides a unique ability to combine multiple functions into a two-component complex that becomes activated in a tumor microenvironment. Therefore, the complex of the present invention provides a significant advantage in having a single method for applying a two-component complex that is located in a tumor microenvironment and can achieve two different signals to benefit a patient. This unique construct provides benefits that are not present in prior art constructs.

Description

[0001] manual

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 780,770, filed December 17, 2018, which is incorporated herein by reference in its entirety for any purpose.

[0004] Sequence Listing

[0005] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled "2019-12-12_01131-0023-00PCT_ST25.txt" created on December 12, 2019, which is 212,992 bytes in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety. Technical Field

[0006] The present application relates to improved targeted immune cell engaging antibody complexes for treating conditions characterized by the presence of cancer cells. In particular, it relates to agents that can be used to treat conditions characterized by the presence of cancer cells. Background Art

[0007] Cancer causes significant loss of life, suffering, and economic impact. Immunotherapeutic strategies to target cancer have become an active area of ​​translational clinical research.

[0008] Various immuno-oncology approaches have been developed to address cancer and other diseases caused by the presence of unwanted cells. In one instance, targeted T cell engaging antibody complexes have shown promising data. See US 10,035,856. These masked compositions comprise a first component and a second component, both of which bind to unwanted cells, and after the inert binding partner is released from each component in the tumor microenvironment, each becomes available to bind to the other. After the first component and the second component bind to each other, they form an active T cell binding domain.

[0009] Other developments have shown that other immune interventions (except for activating T cells) have benefited patients with cancer and other diseases. For example, PD-L1 pathway inhibition can block immune evasion and reverse T cell exhaustion in some tumors (see Chen DS et al. Clin Cancer Res. 18: 6580-6587 (2012)). Simultaneous activation of T cells and binding to tumor-associated antigens such as checkpoint molecules have been proposed as a more robust and selective method for activating T cells against cancer cells (see Harris et al. Cancer Biol Med 13 (2): 171-193 (2016) and Kobold et al. Front Oncol 8: 285 (2018)). However, no single treatment method has included all of these features in a way that various functions become active only at the desired treatment site.

[0010] Therefore, depending on the disease and the patient, further immuno-oncology or other biological interventions may also benefit the patient. Some previous constructs were able to activate T cells in the tumor microenvironment, but did not possess any other properties. Specifically, previous constructs that restricted activity to the desired therapeutic site did not have any way to incorporate additional functional domains. Therefore, additional development in this area of ​​redirected immunotherapy is needed. Summary of the Invention

[0011] The TWICE complexes of the present invention offer the unique ability to combine multiple functions into a two-component complex that becomes activated in the tumor microenvironment. Thus, the complexes of the present invention offer significant advantages in having a single method for administering a two-component complex that is targeted to the tumor microenvironment and can achieve two distinct signals to benefit the patient. This unique construct offers benefits not present in prior art constructs.

[0012] According to the description, the present application describes a kit or composition for treating cancer in a patient, the kit or composition comprising a first component, the first component comprising a targeted immune cell binding agent, the targeted immune cell binding agent comprising a first targeting portion that binds to a tumor antigen expressed by the cancer; a first immune cell binding domain capable of having immune cell binding activity when bound to a second immune cell binding domain, wherein the second immune cell binding domain is not part of the first component, and wherein the first immune cell binding domain is a VH domain or a VL domain; and a first complementary binding domain capable of binding to a complementary antigen when bound to a second complementary binding domain, wherein the second complementary binding domain is not part of the first component, when the first immune cell binding domain is a VH domain, the first complementary binding domain is a VL domain, when the first immune cell binding domain is a VL domain, and wherein the first complementary binding domain is a binding partner of the first immune cell binding domain, such that unless the first immune cell binding domain does not bind to the first complementary domain, the first complementary binding domain binds to the complementary antigen. the first immune cell binding domain does not bind to the second immune cell binding domain; and a second component comprising a targeting immune cell binding agent comprising a second targeting moiety; a second immune cell binding domain capable of having immune cell binding activity when bound to the first immune cell binding domain, wherein if the first immune cell binding domain is a VL, then the second immune cell binding domain is a VH, and wherein if the first immune cell binding domain is a VH, then the second immune cell binding domain is a VL; and a second complementary binding domain capable of binding to a complementary antigen when bound to the first complementary binding domain, wherein when the second immune cell binding domain is a VH domain, the second complementary binding domain is a VL domain, when the second immune cell binding domain is a VL domain, and wherein the second complementary binding domain is a binding partner of 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 second immune cell binding domain does not bind to the second complementary domain.

[0013] In some embodiments, the first immune cell binding domain is bound to the first complementary binding domain through a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the first immune cell binding domain through a first linker; the second dimerization domain is connected to the first complementary binding domain through a second linker; and the first linker and / or the second linker is a cleavable linker. In some embodiments, the first linker and the second linker are cleavable linkers.

[0014] In some embodiments, the second T cell binding domain is bound to the second complementary binding domain through a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the second T cell binding domain through a first linker; the second dimerization domain is connected to the second complementary binding domain through a second linker; and the first linker and / or the second linker is a cleavable linker.

[0015] In some embodiments, the first joint and the second joint are cleavable joints. In some embodiments, the first joint and the second joint are identical. In some embodiments, the first joint and the second joint are different. In some embodiments, the first joint and the second joint are 5 to 30 amino acids in length. In some embodiments, the first joint and the second joint are 8 to 16 amino acids in length. In some embodiments, the protease cleavage site of the first cleavable joint and / or the second cleavable joint is cleaved by a protease expressed by cancer or tumor microenvironment cells. In some embodiments, the protease cleavage site of the first cleavable joint and / or the second cleavable joint is cleaved by a protease that is co-localized to the cancer by a targeting portion that is an antibody or its antigen-binding fragment, the targeting portion is in conjunction with a tumor antigen expressed by the cancer, and is the same or different from the targeting portion in at least one of the first component or the second component.

[0016] In some embodiments, the first dimerization domain and the second dimerization domain are both leucine zippers; immunoglobulin domains; or T cell receptor (TCR) domains. In some embodiments, the immunoglobulin domains comprise immunoglobulin variable domains or immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise CH1 / CL, CH2, CH3 or CH4. In some embodiments, the TCR domain comprises a TCR constant domain. In some embodiments, the dimerization domain in the first component is the same as the dimerization domain in the second component. In some embodiments, the dimerization domain in the first component is different from the dimerization domain in the second component.

[0017] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to cancer when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to an immune checkpoint molecule, RANK or RANKL, or a cell death-inducing antigen when bound to each other.

[0018] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to an immune checkpoint molecule when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to PD-L1 when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of atezolizumab, durvalumab, or avelumab.

[0019] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to RANK when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to RANKL when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of denosumab.

[0020] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to a cell death-inducing antigen when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER, DR6, or CAR1 when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to TRAIL-R1 / DR4 when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of mapatumumab. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of conatumumab (AMG655), lexatumumab, tigatuzumab (CS1008), or drozitumab (PRO95780).

[0021] In some embodiments, the first supplemental binding domain and the second supplemental binding domain are capable of binding to a molecule associated with the extracellular matrix when associated with each other.

[0022] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to a T cell, a macrophage, or a natural killer cell when associated with each other.

[0023] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to T cells when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD3, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 (GITR or TNFRSF18), T cell immunoreceptor with Ig and ITIM domains (TIGIT), or inducible T cell co-stimulator (ICOS) when bound to each other.

[0024] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD3 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of muromonab, otelixizumab, teplizumab, visilizumab, foralumab, SP34, or blinatumomab.

[0025] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to PD-1 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of pembrolizumab or nivolumab.

[0026] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CTLA-4 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of ipilimumab.

[0027] In some embodiments, the first and second supplementary binding domains are capable of binding to TIM-3 when bound together. In some embodiments, the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of TSR-022 or Sym023.

[0028] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to LAG-3 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of BMS-986016.

[0029] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to a KIR when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of lirilumab.

[0030] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD28 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of theralizumab.

[0031] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD 137 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of utomilumab or urelumab.

[0032] In some embodiments, the first supplemental binding domain and the second supplemental binding domain are capable of binding to OX40 when bound together. In some embodiments, the first supplemental binding domain and the second supplemental binding domain comprise all or a portion of the VH and / or VL of PF-04518600 or BMS 986178.

[0033] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD27 when bound together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of varlilumab.

[0034] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to GITR (TNFRSF18) when taken together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of GWN323 or BMS-986156.

[0035] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to TIGIT when taken together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of OMP-313M32, MTIG7192A, BMS-986207, or MK-7684.

[0036] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding ICOS when combined together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of JTX-2011.

[0037] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to macrophages when associated with each other.

[0038] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CSF1R when associated with each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of emactuzumab or IMC-CS4.

[0039] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding CD40 when associated with each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of CP-870,893.

[0040] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to natural killer cells when associated with each other.

[0041] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD 16A when associated with each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of NTM-1633 or AFM13.

[0042] In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to the same antigen.

[0043] In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to different antigens on the same cell.

[0044] In some embodiments, the first supplemental binding domain and the second supplemental binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to different cells.

[0045] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of forming an Fv when not bound to the first supplementary binding domain and the second supplementary binding domain. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of forming an Fv when not bound to the first immune cell binding domain and the second immune cell binding domain.

[0046] The present application also describes a kit or composition for treating cancer in a patient, the kit or composition comprising a first component, the 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; a first immune cell binding domain capable of having immune cell binding activity when bound to a second immune cell binding domain, wherein the second immune cell binding domain is not part of the first component, and wherein the first immune cell binding domain is a VH domain or a VL domain; a first inert binding partner for the first immune cell binding domain, wherein the first inert binding partner 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, wherein if the first immune cell binding domain is a VH domain, then the inert binding partner is a VL domain, and if the first immune cell binding domain is a VH domain, then the inert binding partner is a VL domain, and if the first the immune cell binding domain is a VL domain, then the inert binding partner is a VH domain; a protease cleavage site separating the first immune cell binding domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease, the protease being expressed by the cancer or tumor microenvironment cells; or co-localized to the cancer via a targeting portion that is an antibody or antigen-binding fragment thereof, the targeting portion binding (a) to a tumor antigen expressed by the cancer and being the same or different as the first targeting portion and / or the second targeting portion in the agent, or binding (b) to an antigen expressed by cells in the tumor microenvironment; and a first supplementary functional domain capable of immune cell binding, and a second component comprising a targeted immune cell binding agent comprising a second targeting portion; a second immune cell binding domain; and optionally a second supplementary functional domain capable of immune cell binding.

[0047] In some embodiments, the second component comprises a supplementary functional domain. In some embodiments, the supplementary functional domain of the first component and / or the second component comprises a ligand of a receptor. In some embodiments, the supplementary functional domain is a latent form of a member of the TGF-β family. In some embodiments, the supplementary functional domain of the first component and / or the second component comprises a cytokine. In some embodiments, the cytokine is a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

[0048] In some embodiments, the supplementary functional domain of the first component and / or the second component comprises an attenuated cytokine. In some embodiments, 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.

[0049] In some embodiments, the second component further comprises a second inert binding partner for 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, wherein if the second immune cell binding domain is a VH domain, the inert binding partner is a VL domain, and if the second immune cell binding domain is a VL domain, the inert binding partner is a VH domain; and further, wherein a protease cleavage site separates the second immune cell binding domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease that is expressed by the cancer; or is co-localized to the cancer via a targeting moiety that is an antibody or antigen-binding fragment thereof, which targeting moiety binds (a) a tumor antigen expressed by the cancer and is the same or different from the first targeting moiety and / or second targeting moiety in the agent, or binds (b) an antigen expressed by cells in the tumor microenvironment.

[0050] In some embodiments, the first component is not covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component via a cleavable linker.

[0051] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to a T cell, a macrophage, or a natural killer cell when associated with each other.

[0052] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to T cells when bound to each other. In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD3, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS) when bound to each other.

[0053] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD3 when bound to each other. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of muromonaber, otelizumab, telizumab, vesilizumab, foralizumab, SP34, or blinatumomab.

[0054] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to a T cell receptor when associated with each other.

[0055] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to PD-1 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of pembrolizumab or nivolumab.

[0056] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CTLA-4 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of ipilimumab.

[0057] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to TIM-3 when combined together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of TSR-022 or Sym023.

[0058] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to LAG-3 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of BMS-986016.

[0059] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to a KIR when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of lirelizumab.

[0060] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD28 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of cilalizumab.

[0061] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD 137 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of utomilumab or usulelumab.

[0062] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to OX40 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of PF-04518600 or BMS 986178.

[0063] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD27 when bound together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of varizumab.

[0064] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to GITR (TNFRSF18) when taken together. In some embodiments, the first supplemental binding domain and the second supplemental binding domain comprise all or a portion of the VH and / or VL of GWN323 or BMS-986156.

[0065] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to TIGIT when taken together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of OMP-313M32, MTIG7192A, BMS-986207, or MK-7684.

[0066] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding ICOS when combined together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of JTX-2011.

[0067] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to a macrophage when associated with each other.

[0068] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CSF1R when bound to each other. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of ematuruzumab or IMC-CS4.

[0069] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD40 when bound to each other. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of CP-870,893.

[0070] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to natural killer cells when bound to each other. In some embodiments, the first immune cell binding domain and the second immune binding domain are capable of binding to CD16A when bound to each other.

[0071] In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of NTM-1633 or AFM13. In some embodiments, the first targeting moiety and the second targeting moiety are different.

[0072] In some embodiments, the first targeting moiety and the second targeting moiety are the same.

[0073] In some embodiments, the first targeting moiety and / or the second targeting moiety comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the first targeting moiety and / or the second targeting moiety comprises a DNA aptamer, an RNA aptamer, an albumin, an apolipoprotein, a fibronectin, an ankyrin, a fynomer, an Obody, a DARPin, a knotin, an avimer, an atrimer, an anti-callin, an affilin, an affibody, a bicyclic peptide, a cys knot, an FN3 (adnectin, centryrin, pronectin or TN3) or a Kunitz domain.

[0074] In some embodiments, the second targeting moiety binds to a tumor antigen expressed by the cancer. In some embodiments, the first targeting moiety and / or the second targeting moiety comprises an antibody or antigen-binding fragment thereof that binds to: α4-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 (Synovin); ndecan1); 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; Claudin-1 (CLDN1); CLDN18 (including CLDN18.2); 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; epithelial sialoprotein; FAP; FGFR1; FGFR2; FGFR3; FGFR4; fibronectin additional structure Domain B; FLT-3; flt4; folate receptor 1; guanylyl cyclase C (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 C); TPBG; TRAIL-R1 / DR4; TROP-2; TWEAKR; TYRP1; VANGL2; VEGF; VEGF-C; VEGFR-2; or VEGF-R2. .

[0075] In some embodiments, the first targeting moiety and / or the second targeting moiety comprises an anti-α4-integrin antibody; an anti-CD137 antibody; an anti-CCR4 antibody; an anti-CD123 antibody; an anti-CD133 antibody; an anti-CD138 antibody; an anti-CD19 antibody; an anti-CD20 antibody; an anti-CD22 antibody; an anti-CD33 antibody; an anti-CD38 antibody; an anti-CD40 antibody; an anti-CD49d antibody; an anti-CD52 antibody; an anti-CD70 antibody; an anti-CD74 antibody; an anti-CD79b antibody; an anti-CD80 antibody; an anti-CEA antibody; an anti-cMet antibody; an anti-Cripto antibody; an anti-CTLA-4 antibody; an anti-DLL3 antibody; an anti-TRAIL-2 / DR5 antibody; an anti-E-cadherin antibody; an anti-endoglin antibody; an anti-EpCAM antibody. Antibodies; anti-epidermal growth factor receptor antibodies; anti-FGFR3 antibodies; anti-fibronectin extra domain B antibodies; anti-folate receptor 1 antibodies; anti-glypican 3 antibodies; anti-gp95 / 97 antibodies; anti-Her2 antibodies; anti-IGF-1R antibodies; anti-IL-13R antibodies; anti-IL-4 antibodies; anti-IL-6 antibodies; anti-MMP-9 antibodies; anti-MUC1 antibodies; anti-mucin core protein antibodies; anti-NGcGM3 antibodies; anti-P-cadherin antibodies; anti-PD-L1 antibodies; anti-p-glycoprotein antibodies; anti-PSCA antibodies; anti-PSMA antibodies; anti-SLAMF7 antibodies; anti-TRAIL-R1 / DR4 antibodies; anti-transferrin antibodies; anti-TROP-2 antibodies; or anti-VEGF antibodies or antigen-binding fragments thereof.

[0076] In some embodiments, the first targeting moiety and / or the second targeting moiety comprises 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, L19IL2, L19TNF, Margetuximab, Mirvetuximab, Mogamuizumab, Moxetumomab, Natalizumab, Necitumumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Oportuzumab, Panitumumab nitumumab), PDR001, pembrolizumab, pertuzumab, polatuzumab, racotumomab, ramucirumab, rituximab, rovalpituzumab, sacituzumab, SM3, TAK-164, tositumomab, trastuzumab, tremelimumab, ublituximab, uselumab, utomiliumab, XMAB-5574, or zolbetuximab.

[0077] In some embodiments, the first targeting moiety and / or the second targeting moiety comprises 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. In some embodiments, the first targeting moiety and / or the second targeting moiety comprises the full-length sequence of 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. In some embodiments, the first targeting moiety and / or the second targeting moiety comprises a truncated form, analog, variant, or derivative of 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.

[0078] In some embodiments, the first targeting moiety and / or the 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.

[0079] In some embodiments, the second targeting moiety binds to an antigen expressed by a tumor microenvironment cell. In some embodiments, the tumor microenvironment cell is a fibroblast or a macrophage. In some embodiments, the antigen expressed by a fibroblast is fibroblast activation protein. In some embodiments, the antigen expressed by a macrophage is MAC-1 / CD11b or sideroflexin 3.

[0080] The application also describes a method for treating a cancer that expresses a tumor antigen that binds to a first targeting moiety and / or a second targeting moiety in a patient, the method comprising administering a composition to the patient. In some embodiments, the presence of infiltrating immune cells in the cancer is assessed before administering the composition. In some embodiments, the presence of tumor antigens in the cancer is assessed before administering the composition. In some embodiments, the first targeting moiety and the second targeting moiety bind to the same antigen. In some embodiments, the first targeting moiety and the second targeting moiety bind to different antigens.

[0081] In some embodiments, the cancer that expresses a tumor antigen that binds to the first targeting moiety and / or the second targeting moiety is any of the following: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease, or a premalignant disease.

[0082] In some embodiments, a method of targeting an immune cell to a cancer in a patient that expresses a tumor antigen that binds both a first targeting moiety and / or a second targeting moiety comprises administering a composition to the patient. In some embodiments, a method of targeting an immune cell to a cancer in a patient that expresses two tumor antigens comprises administering a composition to the patient, wherein one tumor antigen binds a first targeting moiety and one tumor antigen binds a second targeting moiety.

[0083] In some embodiments, the method of delivering cytokines to immune cells of a patient comprises administering a composition to the patient, wherein the first supplementary functional domain and / or the second supplementary functional domain of the composition comprises a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

[0084] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0085] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0086] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more implementations and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1A to Figure 1B Double immune cell engager ("TWICE") is shown. Figure 1A Both constructs are shown prior to administration to patients. Figure 1B After being shown in TWICE two components are all targeted to cancer cells, TWICE combines and connects two signaling molecules on an effector (immune) cell. Hexagons and rhombuses represent two different antigens that are combined by the targeting portion (each having two sets of four striped ovals) of the first component and the second component on cancer cells. Each component comprises an immune cell binding domain (the black domain of each component) and a complementary binding domain (the white domain of each component). The joint between the targeting portion and the immune cell binding domain and the complementary binding domain is also shown. When the two components are in close proximity, domain exchange can occur so that two immune cell binding domains (from an immune cell binding domain of each component) and two complementary binding domains (from a complementary binding domain of each component) can be paired with each other. Triangles and circles represent different antigens that can be combined by the paired immune cell binding domain (in conjunction with the antigen represented by the triangle) or the paired complementary binding domain (in conjunction with the antigen represented by the circle) on the effector cell. In the absence of domain exchange and pairing, the immune cell binding domain and the complementary binding domain of the two components do not regulate the effector cell.

[0088] Figure 2Shown is TWICE, which connects two signaling molecules on an effector (immune) cell after the first component is targeted to cancer cells and the second component is targeted to non-cancerous cells. The hexagon represents the antigen bound by the targeting portion (four striped ovals) of the first component on cancer cells. The square represents the antigen bound by the targeting portion (four striped ovals) of the second component on non-cancerous cells (such as tumor-associated macrophages or fibroblasts) in the tumor microenvironment. Each component comprises an immune cell binding domain (the black domain of each component) and a complementary binding domain (the white domain of each component). When the two components are in close proximity, domain exchange can occur so that the two immune cell binding domains (one immune cell binding domain from each component) and the two complementary binding domains (one complementary binding domain from each component) can be paired with each other. The triangle and circle represent different antigens that can be bound by the paired immune cell binding domain (antigen represented by triangle) and the paired complementary binding domain (antigen represented by circle) on the effector cell. In the absence of domain swapping and pairing, the two component immune cell binding domains and complementary binding domains do not modulate effector cells.

[0089] Figure 3Figure 1 shows TWICE binding and bridging signaling molecules on two different effector (immune) cells after both components of TWICE are targeted to cancer cells. Various targeting strategies can be used. If TWICE has two components that bind to cancer cells, it can adopt three different configurations: (a) the two components of TWICE can use two copies of the same targeting moiety to bind to the same antigen (single antigen targeting); (b) TWICE can use a single antigen strategy that utilizes different targeting moieties that target different epitopes on the same antigen; or (c) when both targeting moieties bind to cancer cells, the two components of TWICE can bind to two different antigens (dual antigen targeting). This figure represents single antigen targeting using different targeting moieties that target different epitopes on the same antigen. Returning to this figure, the diamonds represent two molecules of the same antigen on cancer cells that are bound by the targeting moieties of the first component and the second component (two sets with four striped ovals). Each component comprises an immune cell binding domain (the black domain of each component) and a complementary binding domain (the white domain of each component). When the two components are in close proximity, domain swapping can occur so 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 antigens on two different effector cells that can be bound by the paired immune cell binding domains (binding to the antigen represented by the triangle on effector cell number 1) or the paired complementary binding domains (binding to the antigen represented by the circle on effector cell number 2). In the absence of domain swapping and pairing, the immune cell binding domains and complementary binding domains of the two components do not modulate effector cells.

[0090] Figure 4 Figure 20 is a schematic diagram of a TWICE-based approach to target cancer cells. The two components of TWICE are shown to be targeted to cancer cells, binding to and bridging the signaling molecules on effector (immune) cells, and binding to TWICE signaling molecules on cancer cells. Diamonds represent two molecules bound by the targeting moieties of the first and second components (two sets with four striped ovals) of the same antigen on cancer cells. Each component comprises an immune cell binding domain (a black domain for each component) and a complementary binding domain (a white domain for each component). When the two components are in close proximity, domain exchange can occur so that two immune cell binding domains (one immune cell binding domain from each component) and two complementary binding domains (one complementary binding domain from each component) can be paired with each other. Triangles represent antigens that can be bound by paired immune cell binding domains on effector cells. Striped rectangles represent antigens that can be bound by paired complementary binding domains on cancer cells. In the absence of domain exchange and pairing, immune cell binding domains and complementary binding domains do not regulate effector cells or cancer cells.

[0091] Figure 5 Shown are TWICE comprising functional domains before being administered to a patient. The targeting moiety of the first component and the second component (each having two sets of four striped ovals) is shown. Each component comprises an immune cell binding domain (the black curved domain of each component). Each component also comprises an inert binding domain (the white curved domain of each component, also referred to as an inert binding partner) connected to the immune cell binding domain. A component also comprises a supplementary functional domain (black triangle). The joint between the targeting moiety and the immune cell binding domain or the supplementary functional domain is also shown. In addition, the joint between the immune cell binding domain and the inert binding partner of each component is shown. The joint connecting the inert binding partner can be a cleavable joint that is cracked in the tumor microenvironment. This cracking releases the inert binding partner.

[0092] In some embodiments, the two components are closely adjacent to each other and the inert binding partner is released. When the two components are closely adjacent to each other and the inert binding partner is released, domain exchange can occur so that two immune cell binding domains (from an immune cell binding domain of each component) can be paired with each other. In addition, the complementary functional domain can be based on the targeting moiety so that it is positioned to the target cell (such as cancer cells or effector cells) and combines with and connects the target cell. The activity of the complementary functional domain does not need to occur under the pairing with another domain, but is enhanced when the complementary functional domain is positioned to the target cell by the targeting moiety. In the absence of domain exchange and pairing, the immune cell binding domain of the two components does not regulate the target cell. In some embodiments, both components can include the complementary functional domain. In some embodiments, only one component includes the inert binding partner.

[0093] Figure 6TWICE comprising dimerization domain is shown before being applied to the patient.The targeting moiety of the first component and the second component (two sets of four striped ovals are shown).Each component comprises immune cell binding domain (black domain of each component) and complementary binding domain (white domain of each component).The joint between targeting moiety and immune cell binding domain and complementary binding domain is also shown.Dimerization domain is connected to the immune cell binding domain and complementary binding domain (so that each component has associable dimerization domain A and dimerization domain B) of each component by joint.The association of a pair of dimerization domains (pairing of dimerization domain A and dimerization domain B) strengthens the association of the VH domain and the VL domain of each component.Dimerization domain joint can be cleavable, so that after the cracking in tumor microenvironment, joint is cracked, and dimerization domain is lost. After the dimerization domain is removed and when the two components are in close proximity, domain exchange can occur so that two immune cell binding domains (one immune cell binding domain from each component) and two complementary binding domains (one complementary binding domain from each component) can pair with each other. In the absence of domain exchange and pairing, the immune cell binding domains and complementary binding domains of the two components do not regulate target cells. In the absence of cleavage of the dimerization domain linker, the VH domain and VL domain of each component remain united together without domain exchange. In some embodiments, only one component comprises a pair of dimerization domains, while the other component does not comprise a dimerization domain.

[0094] 7A to 7C Expression and cleavage of TWICE molecules with various dimerization domain linkers and dimerization domains listed in Table 12 are shown. Figure 7A TWICE with various dimerization domain linkers and dimerization domains are shown. Figure 7B ) were analyzed TWICE, and the cleavage products ( Figure 7C ).

[0095] Figures 8A to 8B It is shown that TWICE containing Fab or dsFv binding moieties undergoes a range of concentrations ( Figure 8A ) or EC50 measurement results ( Figure 8B ) target binding achieved.

[0096] Figures 9A to 9C Bridging ELISA showing CD3 / CTLA4 TWICE with hetero-Fc dimerization domain. Figure 9A Shows assay design, while Figure 9B Results obtained with CD3 bridging are shown, and Figure 9CResults obtained with CTLA-4 bridging are shown.

[0097] FIG. 10A to FIG. 10B The CD3 / CTLA4 TWICE with coiled-coil dimerization domain was shown to be a CD3 bridging ELISA ( Figure 10A ) or CTLA4 bridging ELISA ( Figure 10B ) results obtained.

[0098] Figures 11A to 11B Showing CD28 / CD3 TWICE in CD3 bridging ELISA ( Figure 11A ) or CD28 bridging ELISA ( Figure 11B ) in the result.

[0099] FIG. 12A to FIG. 12B A bridging ELISA with Cilalizumab (anti-CD28) and Urelizumab (anti-4-1BB) TWICE is shown. Specifically, this shows that CD28 / 4-1BB (CD137) TWICE is effective in the CD28 bridging ELISA ( Figure 12A ) or 4-1BB bridging ELISA ( Figure 12B ) in the result.

[0100] Figure 13 Showing PD-L1 binding of atezolizumab / ipilimumab TWICE. Specifically, this shows the results of PD-L1 / CTLA-4 TWICE in a PD-L1 bridging ELISA.

[0101] FIG. 14A to FIG. 14B Shown is T cell redirection on HCT-15 cells achieved by CD3 / CTLA4 TWICE with or without pre-lysis. Figure 14A Activation by pre-cleavage is shown. Figure 14B Results from experiments with and without pre-lysis are shown.

[0102] 15A to 15D Demonstrating T cell redirection and HCT15 cell killing achieved by CD3 / CTLA-4 TWICE. Figure 15A Results of cleavage analysis are shown. Figure 15B Shows interferon gamma release. Figure 15C and Figure 15D Shown are the results of measuring specific lytic LDH release from HCT15 cells under different conditions.

[0103] Figure 16 Shows T cell redirection on HCT-15 achieved by CD3 / CD28 TWICE.This figure shows HCT-15 cell killing achieved by CD3 / CD28 TWICE as measured by LDH release.

[0104] 17A to 17B Redirection of preactivated / exhausted peripheral blood mononuclear cells (PMBCs) to HCT15 cells was demonstrated as determined by flow cytometry ( Figure 17A ) or by interferon gamma release ( Figure 17B ) measured.

[0105] 18A to 18B NK cell redirection against HCT15 cells was demonstrated as measured by CD16a bridging ELISA ( Figure 18A ) or released by LDH ( Figure 18B ) measured.

[0106] Sequence Description

[0107] Table 1 provides a listing of certain sequences cited herein.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] DETAILED DESCRIPTION

[0139] I. Dual immune cell adapter

[0140] A dual immune cell engager ("TWICE") refers to a two-component complex in which the domains of the two components can pair and bind and engage immune cells. Thus, a single TWICE comprises two components that can be present in two separate polypeptides or in a single polypeptide. Thus, a TWICE can be a single construct, or it can be a kit with two components. Unless explicitly discussed otherwise, the use of the term TWICE throughout the application and claims includes both embodiments. This application describes a series of TWICEs. TWICEs can activate different pathways to mediate multiple anti-cancer effects.

[0141] As will be described in this application, TWICE may include a first component and a second component, each of which includes a targeting moiety, an immune cell binding domain and a complementary binding domain. The targeting moiety of the first component and the second component serves to target TWICE to cells in cancer cells or tumor microenvironment. When the immune cell binding domains from the two components are paired together and / or the complementary binding domains from the two components are paired together, these paired domains can play a role in regulating immune cell activity, or play other (i.e., complementary) functions. For example, the complementary binding domains of the pairing can block immune checkpoints or induce cell death of cancer cells.

[0142] The presence of the complementary binding domains in TWICEs is expected to improve their efficacy in treating cancer compared to complexes that do not comprise the complementary domain (ie, compared to complexes that bind only a single antigen on immune cells).

[0143] As will be described herein, the first component, and optionally the second component, may comprise a complementary functional domain capable of immune cell binding. In some embodiments with a complementary functional domain, the immune cell binding domain is masked. The complementary functional domain of TWICE can modulate immune function without the need for pairing with a different domain.

[0144] A major advantage of the TWICE complexes of the invention described herein is that the immune cell binding domains, and in some embodiments, the complementary binding domains, will only pair when the first and second components of TWICE are both in close proximity (here, in the tumor microenvironment, since they are both targeted to cancer). At the cancer site, domain pairing between the different components of TWICE can improve specificity and reduce off-target effects because the immune cell binding domains, and optionally, the complementary binding domains, only function after the domains from both components pair at the cancer cell or its microenvironment.

[0145] After describing some representative types of TWICE, this application will then describe the components that make up the different types of TWICE. TWICE is not limited to the representative descriptions herein, as TWICE can modulate many pathways related to cancer survival and anti-cancer immune responses.

[0146] The TWICE complex of the present invention provides a unique ability to combine multiple functions into a two-component complex that becomes activated in a tumor microenvironment. Therefore, the complex of the present invention provides a significant advantage in having a single method for administering a two-component complex that is localized to the tumor microenvironment and can achieve two different signals to benefit the patient. In some embodiments, one of those signals (from the paired immune cell binding domain) is activated only when the two-component complex meets. In some embodiments, both of those signals (from the paired immune cell binding domain and from the paired complementary binding domain) are activated only when the two-component complex meets. This unique construct provides many benefits that are not present in prior art constructs.

[0147] 1. Activating two signaling pathways twice on one type of immune cell

[0148] In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, bind the same type of immune cell.

[0149] As used herein, immune cells may also be referred to as “effector cells.” The term “effector cells” means immune cells that mediate activity against cancer.

[0150] In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, bind to T cells.

[0151] In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, bind to the same antigen on the immune cell. In this embodiment, the two signal transduction pathways are identical, and the effect is amplified. In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, bind to different antigens on the immune cell. In this embodiment, the two signal transduction pathways are different, and two different signals are transmitted to the immune cell.

[0152] In general, the process of T cell activation involves a variety of signals. Primary signal comes from the combination of T cell receptor (TCR) and major histocompatibility complex (MHC) molecules presented by antigen presenting cells (APC). Co-stimulatory signals can be produced by one of several different T cells and APC interactions (see Buchbinder EI, Desai A Am J Clin Oncol. 39 (1): 98-106 (2016)). The activation of T cells is enhanced by several co-stimulatory receptors, such as CD28, CD137 (4-1BB), OX40, CD27, GITR (TNFRSF18) and ICOS. The interaction between T cells and APC or cancer cells can also be related to several inhibitory / checkpoint molecules that reduce T cell activation, such as PD-1, CTLA-4, TIM-3, LAG-3 and KIR (Torphy RJ et al. Int J Mol Sci. 18: 2642 (2017)). Therefore, combining the effects of two antibodies directed against co-stimulatory or co-inhibitory receptors can enhance anti-tumor T cell responses.

[0153] TWICE can contain (after pairing) two functional antibodies against co-stimulatory or checkpoint molecules on T cells, resulting in robust T cell activation ( Figure 1B , showing pairing of two component immune cell binding domains in black and two component complementary binding domains in white). Robust T cell activation can be used in patients when T cells are present in tumors but are inactive due to immunosuppressive signals from cancer cells (Wu AA et al. OncoImmunology 4:7 (2015)).

[0154] In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, can engage immune cells through agonistic or antagonistic binding to T cells.

[0155] By "agonistic," it is meant that binding of the paired immune cell binding domains or paired complementary binding domains to the antigen activates a signaling pathway. Agonistic binding may be of interest when binding to the antigen results in immune cell activation.

[0156] By "antagonistic," it is meant that the binding of a paired immune cell binding domain or a paired complementary binding domain to an antigen blocks or inhibits a signaling pathway. Antagonistic binding may be of interest when binding of a natural ligand to an antigen normally leads to immune cell deactivation. In this way, binding of a set of "antagonistic" paired domains of the present invention's TWICE can activate immune cells by blocking or inhibiting pathways that normally mediate immune cell anergy or deactivation.

[0157] Antibodies known to have antagonistic or agonistic effects on immune cells can be used to generate paired binding domains for the TWICE of the present invention. If a signaling pathway promotes T cell activation, then an agonistic approach will increase T cell activation. If a signaling pathway inhibits T cell activation, then an antagonistic approach will increase T cell activation. Therefore, many different types of pathways can be used in this approach.

[0158] An example of an agonistic antibody is a CD3ε antibody (e.g., SP34, OKT3) that stimulates the TCR complex. In another example, an agonistic antibody against GITR can activate CD8+ T effector cells while inhibiting regulatory T cell function (see Knee et al., European Journal of Cancer 67: 1e10 (2016)). An example of an antagonistic antibody that causes stimulation of immune cells is a PD-1 antibody (e.g., pembrolizumab or nivolumab). PD-1 inhibits immune cells, and therefore, these antibodies block the interaction of PD-1 with its ligands and thereby antagonize inhibitory signals.

[0159] In some embodiments, when the immune cell binding domains are paired together, and when the complementary binding domains are paired together, two agonistic antibodies can be generated and two positive stimuli can be provided to the immune system (e.g., an anti-CD3ε antibody and an anti-CD137 antibody). Both CD3 and CD137 promote T cell activation.

[0160] In some embodiments, when the immune cell binding domains are paired together, and when the complementary binding domains are paired together, an agonistic antibody and an antagonistic antibody (e.g., an anti-CD3ε antibody and an anti-CTLA4 antibody) can be produced. As discussed above, CD3 stimulates the TCR complex, while CTLA4 provides an inhibitory signal. Antagonizing the inhibitory signal of CTLA4 will promote T cell activation.

[0161] In some embodiments, when the immune cell binding domains are paired together, and when the complementary binding domains are paired together, two antagonistic antibodies (e.g., an anti-CTLA4 antibody and an anti-PD-1 antibody) can be generated that block two inhibitory signals. As discussed above, both of these signals inhibit T cell activation, and antagonizing them will promote T cell activation.

[0162] Another exemplary TWICE combines a CD3ε (CD3e) antibody with another antibody that promotes T cell activation. Current evidence suggests that CD3 bispecific antibodies are further regulated by different checkpoint molecules (see Kobold S et al. Front Oncol. 8: 285 (2018)). In addition, in some patients or environments, the therapeutic effects of T cell activating bispecific antibodies are also limited by induced T cell anergy (absence of a normal immune response to a specific antigen). Therefore, combining an anti-CD3ε antibody that activates T cells with an antibody that enhances T cell activation and prevents anergy can produce enhanced and / or more persistent anti-tumor T cell responses.

[0163] 2. Twice: Regulating Two Types of Immune Cells

[0164] In some embodiments, the immune cell binding domains, when paired together, and the complementary binding domains, when paired together, bind to two different immune cells. In this way, TWICE can modulate two different types of immune cells. A wide range of different immune cells can be modulated by TWICE, including the following representative examples. Figure 2 A representative model showing the activation of two immune cells (effector 1 and effector 2) by TWICE.

[0165] a) TWICE stimulates T cells and inhibits tumor-associated macrophages

[0166] The combination of two different immune cells can be used in tumor microenvironments where T cells are excluded from the tumor and tumor-associated macrophages (TAMs) may be present. TAMs represent a class of alternatively activated macrophages that promote tumor growth by promoting angiogenesis, immunosuppression, and inflammation (Poh and Ernst, Front Oncol. 12(8):49(2018)). In some embodiments, when the complementary binding domain and the immune cell binding domain are paired, TWICE combines the activities by blocking TAM activity and activating T cells.

[0167] In some embodiments, TWICE comprises an immune cell binding domain that, when paired together, mediates T cell activation, and a complementary binding domain that, when paired together, inhibits TAMs.

[0168] For example, targeting TAMs with anti-CSF1R blocking antibodies reduces TAM infiltration and promotes CD8+ T cell expansion (Ries CH et al. Cancer Cell. 25(6):846-59(2014)). Activating the CD40 pathway can also interfere with the immunosuppressive effects of TAMs. CD40 agonistic antibodies can lead to the reprogramming or recruitment of classically activated macrophages, which can be used to trigger an effective immune response against tumors. Following pairing of the immune binding domain and the complementary binding domain, a representative TWICE combining a T cell activating antibody and a CD40 agonistic antibody or an anti-CSF1R blocking antibody can provide additive efficacy compared to using either of these antibodies alone.

[0169] b) TWICE stimulation of T cells and NK cells

[0170] TWICE can be used to activate both T cells and NK cells to kill cancer cells. Compared to activating either cell alone, co-activating T cells and NK cells can lead to a more robust immune response to cancer.

[0171] In some embodiments, TWICE comprises an immune cell binding domain that, when paired together, mediates T cell activation and a complementary binding domain that, when paired together, mediates NK cell activation, such as that achieved with an anti-CD16A antibody. For example, in TWICE comprising TWICE440 and TWICE441, an anti-CD16A antibody is combined with an anti-CD3 antibody, OKT3 (see SEQ ID NOs: 212-215 and 18A to 18B ).

[0172] 3. TWICE stimulates immune cells and mediates effects on cancer cells

[0173] TWICE can also bind to immune cells using its paired immune cell binding domains and cancer cells using its paired complementary binding domains. This therapeutic strategy can directly induce biological functions in targeted cancer cells and mediate the activation of anti-cancer immune cells. The biological functions induced in cancer cells can, for example, induce cell death via death receptors or inhibit immunosuppressive signaling.

[0174] Paired immune cell binding domains that can activate immune cells have been described, and TWICE components may also contain complementary binding domains that bind cancer.

[0175] a) TWICE: Stimulating immune cells and blocking immune cell deactivation caused by cancer

[0176] The TWICE of the present invention can be used to activate immune cells and block inhibitory signals from cancer cells to immune cells. In some embodiments, the complementary binding domains, when paired together, can block signals from cancer cells that inhibit immune cell activation.

[0177] For example, some cancers express PD-L1, which can bind to PD-1 or B7.1 on T cells and cause T cell inactivation. In some embodiments, the complementary binding domains bind to PD-L1 on cancer cells when paired together. In some embodiments, binding to PD-L1 blocks the interaction with PD-1 on T cells. Blocking the interaction between PD-1 and PD-L1 has been shown to improve anti-tumor responses. PD-L1 antibodies (such as atezolizumab, durvalumab, and avilumab) are approved for the treatment of several malignancies, including non-small cell lung cancer and melanoma.

[0178] In another example, some cancers may express members of the tumor necrosis factor receptor (TNFR) family, which downregulate anti-tumor immune responses. An example of such a molecule is receptor activator of nuclear factor kappa-B (RANK), which binds its ligand RANKL on regulatory T cells to create an immunosuppressive environment (see Wu et al. Oncoimmunology.4(7)(2015)). In some embodiments, the complementary binding domains bind to RANK on cancer cells when paired together. In some embodiments, binding to RANK blocks the interaction with RANKL on T cells. In a preclinical mouse model of cancer, blocking RANK signaling with an antagonistic antibody to RANKL enhanced the anti-tumor effect of anti-CTLA4 antibodies that can activate T cells (Ahern et al. Clin Cancer Res.23(19):5789-5801(2017)).

[0179] b) TWICE stimulates immune cells and triggers cell death in cancer cells

[0180] The TWICE of the present invention can also be used to activate immune cells and trigger cell death of cancer cells. In some embodiments, the complementary binding domains, when paired together, can trigger the death of cancer cells by binding to receptors expressed by cancer cells.

[0181] Examples of receptors that can be stimulated to trigger cell death include members of the TNFR family, such as Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER or CAR1. A TNFR family agonist antibody that induces cell death of cancer cells in clinical development is mapatumumab (NCT01258608). TWICE, which combines the activity of directly killing cancer cells with activating an anti-cancer immune response, may have additive efficacy.

[0182] 4. TWICE containing complementary functional domains

[0183] The first component and / or the second component of TWICE may also include a supplementary functional domain. As used herein, a "supplementary functional domain" refers to a molecule that modulates immune function when targeted to appropriate immune cells without the need for pairing with another domain. Thus, TWICE can target the supplementary functional domain to immune cells and promote the activity of supplementary functional domains that would lack activity or be too toxic to be delivered systemically without targeting. An exemplary supplementary functional domain is an attenuated cytokine that must be targeted to appropriate immune cells to be active. This embodiment is shown in Figure 5 Attenuated cytokines can preferentially activate target cells, as shown by data obtained with bempegaldesleukin (NKTR-214), a CD-122-preferential IL-2 pathway agonist.

[0184] In this way, TWICE can mediate an additive anti-cancer immune response achieved by the activity of the paired immune cell-binding domains as well as activity from complementary functional domains—in effect, delivering a "package of care" to effector cells that is activated only at effector cell / cancer cell junctions.

[0185] B. Single polypeptide chain or two components

[0186] TWICE can comprise a single polypeptide chain or two separate components.

[0187] In some embodiments, the first component is not covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component.

[0188] In some embodiments, TWICE comprises two separate components. In other words, TWICE may comprise a first component and a second component that are separate polypeptides.

[0189] In some compositions, TWICE comprises a single polypeptide chain. In some embodiments, the first component and the second component are contained within a single amino acid sequence.

[0190] When TWICE comprises a single polypeptide chain, the first component and the second component can be separated by a linker. In some embodiments, this linker covalently binds the first component and the second component. In some embodiments, this linker comprises a cleavable linker. In some embodiments, the cleavable linker between the first component and the second component comprises a protease cleavage site.

[0191] In some embodiments, the cleavage site contained in the linker that covalently binds the first component and the second component is a protease cleavage site. SEQ ID NO: 1-84 lists some exemplary protease cleavage sites that can be used, but TWICE is not limited to this group of protease cleavage sites and other protease cleavage sites can be used.

[0192] In some embodiments, the cleavage site contained in the joint that covalently binds the first component and the second component is a tumor-associated protease cleavage site. Tumor-associated protease is a protease associated with a tumor. In some embodiments, relative to other areas of the body, the tumor-associated protease has higher expression in the tumor. Table 2 (shown in the following sections IV) provides examples of tumor-associated proteases, but any protease expressed in the tumor can be used to select the tumor-associated protease cleavage site for TWICE. In some embodiments, the protease is expressed by non-cancerous cells such as tumor-associated macrophages or fibroblasts in the tumor microenvironment.

[0193] In some embodiments, the cleavage site contained within the linker that covalently binds the first component and the second component is a cleavage site of a protease found in blood. Exemplary proteases found in blood include thrombin, neutrophil elastase, and furin.

[0194] C. Targeting Moiety

[0195] The targeting moiety of the first component acts by delivering the agent to the local environment of the cancer cell, thereby enabling a localized treatment strategy. In some embodiments, the first targeting moiety targets the cancer cell by specifically binding to the cancer cell.

[0196] In some embodiments, the first targeting moiety and the second targeting moiety both bind to a tumor antigen expressed by the cancer.

[0197] In some embodiments, the first targeting moiety binds to a tumor antigen expressed by the cancer and the second targeting moiety binds to an antigen expressed by cells of the tumor microenvironment.

[0198] In some embodiments, the targeting moiety is an antibody or its antigen-binding fragment. With respect to antigen-binding fragment, we mean any antibody fragment that retains its binding activity to target, such as scFv or other functional fragments, including immunoglobulins, VHH, VNAR, Fab, Fab', F(ab')2, Fv, antibody fragment, miniature bifunctional antibody, scAB, single domain heavy chain antibody, single domain light chain antibody, Fd, CDR district or antibody that lack light chain and can bind any part or peptide sequence of antigen or epitope. VHH and VNAR are substitutes for classical antibodies, and although they are produced in different species (being respectively camelids and shark), we also include them in the antigen-binding fragment of antibody. In some embodiments, the Fv domain is disulfide bond stabilized Fv (dsFv). Unless clearly indicated as " full-length antibody ", otherwise when the application refers to antibody, it inherently includes the mentioning of its antigen-binding fragment.

[0199] In some embodiments, the targeting moiety is not an antibody, but another type of targeting moiety. A wide range of targeting moieties capable of targeting cancer are known, including DNA aptamers, RNA aptamers, albumin, apolipoprotein, fibronectin, ankyrin, fynomer, Obody, DARPin, knotin, affinity polymer, atrimer, anticallin, affilin, affibody (affibody), bicyclic peptide, cys knot, FN3 (adnectin, centryrin, probent, TN3) and Kunitz domain. These and other non-antibody skeleton structures can be used to target cancer cells. Smaller non-antibody skeletons are quickly removed from the bloodstream and have a shorter half-life compared to monoclonal antibodies. Due to rapid extravasation from the capillary lumen through the vascular endothelium and basement membrane, they also show faster tissue penetration. See Vazquez-Lombardi et al., Drug Discovery Today 20 (1): 1271-1283 (2015). Many non-antibody scaffolds targeting cancer are already in clinical development, with additional candidates in the preclinical stage (see Vazquez-Lombardi, Table 1).

[0200] 1. Targeting moieties capable of targeting cancer

[0201] In some embodiments, only the first targeting moiety binds to an antigen expressed by the cancer, while the second targeting moiety does not. In some embodiments, both the first targeting moiety and the second targeting moiety bind to an antigen expressed by the cancer.

[0202] In some embodiments, the first targeting moiety and the second targeting moiety are the same and bind to the same antigen. In some embodiments, the first targeting moiety and the second targeting moiety bind to the same antigen expressed by the cancer. In some embodiments, the first targeting moiety and the second targeting moiety bind to different epitopes on the same antigen expressed by the cancer. In some embodiments, the first targeting moiety and the second targeting moiety bind to different antigens expressed by the cancer.

[0203] Certain tumor antigens that can be used for targeting (examples of cancer cell types are in parentheses) include: Her2 / Neu (epithelial malignancies); CD22 (B-cell malignancies); EpCAM (CD326) (epithelial malignancies); EGFR (epithelial malignancies); PSMA (prostate cancer); 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 lymphoma); CA125 (various cancers, including ovarian cancer); carbonic anhydrase IX (various cancers, including renal cell carcinoma); CD70 (B-cell malignancies); CD74 (B-cell malignancies); CD56 (T-cell or NK-cell lymphoma); CD40 (B-cell malignancies); CD19 (B-cell malignancies); c-met / HGFR (gastrointestinal and liver malignancies) ); TRAIL-R1 / DR4 (multiple malignancies, including ovarian and colorectal cancer); DRS (multiple malignancies, including ovarian and colorectal cancer); PD-1 (B-cell malignancies); PD-L1 (multiple malignancies, including epithelial adenocarcinomas); IGF-1R (most malignancies, including epithelial adenocarcinomas); VEGF-R2 (vascular structures associated with most malignancies, including epithelial adenocarcinomas); prostate stem cell antigen (PSCA) (prostate adenocarcinoma); MUC1 (epithelial malignancies); CanAg (tumors, such as colon and pancreatic cancer); mesothelin (many tumors, including mesotheliomas and ovarian and pancreatic adenocarcinomas); P-cadherin (epithelial malignancies, including breast adenocarcinoma); myostatin (GDF8) (many tumors, including sarcomas and ovarian and pancreatic adenocarcinomas); Cripto (TDGF1) (epithelial malignancies, including colon, breast, lung, ovarian, and pancreatic cancers); ACVRL 1 / ALK1 (multiple malignancies, including leukemias and lymphomas); MUC5AC (epithelial malignancies, including breast adenocarcinoma); CEACAM (epithelial malignancies, including breast adenocarcinoma); CD137 (B-cell or T-cell malignancies); CXCR4 (B-cell or T-cell malignancies); neuropilin 1 (epithelial malignancies, including lung cancer); glypican (multiple cancers, including liver, brain, and breast cancer); HER3 / ERBB3 (epithelial malignancies); PD GFRa (epithelial malignancies); EphA2 (multiple cancers, including neuroblastoma, melanoma, breast cancer, and small cell lung cancer); CD38 (myeloma); CD138 (myeloma); α4-integrin (AML, myeloma, CLL, and most lymphomas); CCR4 antibody (lymphoma); CD52 (leukemia); CD79b (lymphoma); CTLA-4 (multiple cancers, including non-small cell lung cancer, head and neck cancer, urothelial carcinoma, and hepatocellular carcinoma); DLL3 (lung cancer);Endoglin (multiple cancers, including soft tissue sarcomas, angiosarcomas, and renal cell carcinoma); fibronectin extra domain B (melanoma); folate receptor 1 (multiple cancers, including epithelial ovarian, peritoneal, and fallopian tube cancers); MMP-9 (gastric or gastroesophageal junction adenocarcinoma); NGcGM3 (lung cancer); SLAMF7 (multiple myeloma); TROP-2 (breast cancer); or VEGF (colorectal cancer).

[0204] In some embodiments, the targeting moiety binds to α4-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; C D22; 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; CLDN18.2; 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; epithelial sialoprotein; 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; TWEAKR; TYRP1; VANGL2; VEGF; VEGF-C; VEGFR-2; or VEGF-R2.

[0205] Table 2 (shown in Section IV below) provides non-limiting examples of cancer types, possible targets of targeting moieties, and proteases expressed by those cancer types. Proteases associated with cancer may be referred to as tumor-associated proteases. To prepare a TWICE that needs to be cleaved at the tumor site, the cancer can be identified, and the target of the targeting moiety can be selected (as needed), and one or two proteases of the cancer type or desired tumor microenvironment cells (such as TAM or TAF) can be selected. Not every TWICE requires protease cleavage, but protease cleavage can be beneficial for TWICE produced in the form of a single molecule with a cleavage site or a TWICE comprising an inert binding partner.

[0206] Table 3 (shown in Section IV below) provides additional information about cancers that can be targeted with different targeting moieties, including the fact that some targeting moieties can target many different types of cancer. In TWICE, both the first component and the second component can contain a targeting moiety that can target cancer. The targeting moieties of the first component and the second component can bind to the same antigen or different antigens.

[0207] Antibodies that bind to tumor antigens and are specific for tumor cells are well known in the art.

[0208] FDA maintains a list of approved antibody drugs for the treatment of cancer, many of which are in combination with cancer antigens and can be used in this case. Referring to The Orange Book Online or Drugs@FDA on the FDA website. FDA also maintains a list of ongoing clinical trials in the clinicaltrials.gov database, which can be searched by disease name. Table 4 (shown in Sections IV below) provides a representative list of approved antibodies that are specific to tumor cells. Table 5 (shown in Sections IV below) provides a representative list of antibodies that are specific to tumor cells that are in development.

[0209] Table 6 (shown in Section IV below) summarizes selected publications on exemplary antibodies that bind to tumor antigens and can be used as targeting moieties in TWICE. These publications show that targeting moieties capable of binding to tumor antigens are well within the skill in the art and the inventors' grasp at the time of filing this application.

[0210] Other antibodies well known in the art can be used as targeting moieties for targeting a given cancer. Antibodies and their corresponding antigens include nivolumab (anti-PD-1Ab), TA99 (anti-gp75), 3F8 (anti-GD2), 8H9 (anti-B7-H3), abagovomab (anti-CA-125 (generic)), adecatumumab (anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2), altumomab pentetate (anti-CEA), amatuximab (anti-mesothelin), AME-133 (anti-CD20), anatumomab mafenatox (anti-TAG-72), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA), bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab (anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab (anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab (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, CD3), cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49 (anti-TAG-72), cedelizumab (anti-CD4), Ch.14.18 (anti-GD2), ch-TNT (anti-DNA-associated antigen), citatuzumab bogatox (anti-EpCAM), cixutumumab (anti-IGF-1 receptor), clivatuzumabtetraxetan (anti-MUC1), canagliflozin (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab (anti-CD40), 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), zozituzumab (anti-DR5), duligozumab ( duligotumab (anti-HER3), dusigitumab (anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab (anti-EpCAM), elotuzumab (anti-SLAMF7), elsilimomab (anti-IL-6), enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), ensituximab (anti-5AC), epitumomab (anti-IL-6), cituxetan (anti-epithelial sialoprotein), epratuzumab (anti-CD22), ertumaxomab (anti-HER2 / neu, CD3), etaracizumab (anti-integrin αvβ3), faralimomab (anti-interferon receptor), farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20), ficlatuzumab (anti-CD20), umab (anti-HGF), figitumumab (anti-IGF-1 receptor), flanvotumab (anti-TYRP1 (glycoprotein 75)), fresolimumab (anti-TGFβ), futuximab (anti-EGFR), galiximab (anti-CD80), ganitumab (anti-IGF-I), gemtuzumab ozogamicin (anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CA-IX)), glembatumumab (anti-VEGF-β), and vedotin.vedotin (anti-GPNMB), guselkumab (anti-IL13), ibalizumab (anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1), igovomab (anti-CA-125), zolbetuxin (IMAB362, anti-CLDN18.2), IMC-CS4 (anti-CSF1R), IMC-TR1 (TGFβRII), imgatuzumab (anti-EGFR), inclacumab (anti-selectin P), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin (anti-CSF1R), and tadalafil (anti-CD4). ozogamicin (anti-CD22), intetumumab (anti-CD51), ipilimumab (anti-CD152), iratumumab (anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-0761 (anti-CD194), LY2875358 (anti-MET), labetuzumab (anti-CEA), lambrolizumab (anti-PDCD1), lexalimumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirelizumab (anti-KIR2D), motansin and lorvotuzumab mertansine (anti-CD56), lucatumumab (anti-CD40), lumiliximab (anti-CD23 (IgE receptor)), mapatumumab (anti-TRAIL-R1), magetuximab (anti-ch4D5), matuzumab (anti-EGFR), mavrilimumab (anti-GMCSF receptor α-chain), milatuzumab (anti-CD74), minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside), mogamulizumab (anti-CCR4), moxetumomab pasudotox (anti-CD22), nacolomab tafenatox (anti-C242 antigen), naptumomabestafenatox (anti-5T4), narnatumab (anti-RON), nesvacumab (anti-EGFR), nesvacumab (anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4), nofetumomab merpentan, ocrelizumab (anti-CD20), ocaratuzumab (anti-CD20), oclatuzumab (anti-PDGF-Rα), onartuzumab (anti-c-MET), ontuxizumab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab (anti-CA-125), otlertuzumab (anti-CD37), pankomab (anti-tumor-specific glycosylation of MUC1), parsatuzumab (anti-EGFL7), pascolizumab (anti-IL-4), patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab (anti-HER2 / neu), pidilizumab (anti-PD-1), pinatuzumab vedotin (anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumab vedotin (anti-CD22), vedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20), quilizumab, (anti-IGHE), ratumomab (anti-N-glycolylneuraminic acid), radretumab (anti-fibronectin extra domain-B), ramucirumab (anti-VEGFR2), rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab (anti-RHD), rovelizumab (anti-CD11 and CD18), samalizumab (anti-CD200), satumomab (anti-VEGFR2), pendetol (anti-CD11 and CD18), samalizumab (anti-CD200), satumomab (anti-VEGFR2), rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab (anti-RHD), rovelizumab (anti-CD11 and CD18), samalizumab (anti-CD200), satumomab (anti-VEGFR2), pendetol (anti-CD11 and CD18), samalizumab (anti-CD200), pendetol (anti-CD11 and CD18), pendetol (anti-CD11 and CD18), pendetol (anti-CD20 ...pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A (anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab (anti-IL-6), solitomab (anti-EpCAM), sontuzumab (anti-epithelial sialoprotein), tabalumab (anti-BAFF), tacatuzumabtetraxetan (anti-alpha-fetoprotein), taplitumomab paptox (anti-CD19), telimomab aritox), tenatumomab (anti-tenasin C), teneliximab (anti-CD40), teprotumumab (anti-CD221), TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tegatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20), tovetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4), tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab celmoleukin (anti-EpCAM), utuximab (anti-CD20), usrelizumab (anti-4-1BB), vantictumab (anti-Frizzled receptor), vapaliximab (anti-AOC3 (VAP-1)), vatelizumab (anti-ITGA2), veltuzumab (anti-CD20), vesencumab (anti-NRP1), vesilizumab (anti-CD3), volociximab (anti-integrin α5β1), mavodotetin (vorsetuzumab)

[0015] The following are antibodies against glioma (anti-glioma), glioma (anti-glioma), gliomab (anti-glioma), gliomaf ...

[0211] Antibodies that bind to these antigens can also be used in TWICE, specifically targeting the indicated cancer types: Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancer), CA15-3 (carcinoma), CA19-9 (carcinoma), L6 (carcinoma), Lewis Y (carcinoma), Lewis X (cancer), alpha-fetoprotein (cancer), CA242 (colorectal cancer), placental alkaline phosphatase (cancer), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (cancer), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3ε (T-cell lymphoma, lung cancer, breast cancer, gastric cancer, ovarian cancer, autoimmune diseases, malignant ascites), CD19 (B-cell malignancies), CD20 (non-Hodgkin's lymphoma, B-cell neoplasms, autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, systemic lupus erythematosus), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma), sarcomas), CD52 (leukemias), CD56 (small cell lung cancer, ovarian cancer, Merkel cell carcinoma and liquid tumors, multiple myeloma), CD66e (carcinomas), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (carcinomas), CD123 (leukemias), mucins (carcinomas), CD221 (solid tumors), CD22 (breast, ovarian cancer), CD262 (NSCLC and other cancers), CD309 (ovarian cancer), CD326 (solid tumors), CEACAM3 (colorectal cancer, gastric cancer), CEACAM5 (CEA, CD66e) (breast, colorectal and lung cancer), DLL4 (A-like 4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD 184, hematological cancers, solid tumors), endoglin (CD105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder cancer, head cancer, neck cancer, colon cancer, NHL, prostate cancer, and ovarian cancer), ERBB2 (lung cancer, breast cancer, prostate cancer), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancer), FGFR (cancer), GD2 ganglioside (cancer), G-28 (cell surface antigen glycolipid, melanoma), GD3 idiotype (cancer), heat shock protein (cancer), HER1 (lung cancer, gastric cancer), HER2 (breast cancer, lung cancer, and ovarian cancer), HLA-DR10 (NHL), HLA-DRB (NHL, B-cell leukemia), human chorionic gonadotropin (cancer), IGF1R (solid tumors, blood cancers), IL-2 receptor (T-cell leukemia and lymphoma), IL-6R (multiple myeloma, RA, Castleman's disease disease), IL6-dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, for various cancers), MAGE-1 (cancer), MAGE-2 (cancer), MAGE-3 (cancer), MAGE 4 (cancer), anti-transferrin receptor (cancer), p97 (melanoma), MS4A1 (transmembrane 4 domain subfamily A member 1, non-Hodgkin's B-cell lymphoma, leukemia), MUC1 (breast cancer, ovarian cancer, cervical cancer, bronchial cancer and gastrointestinal cancer), MUC16 (CA125) (ovarian cancer), CEA (colorectal cancer), gp100 (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (transmembrane 4 domain subfamily A, small cell lung cancer, NHL), nucleolin, Neu oncogene product (cancer), p21 (cancer), nectin-4 (cancer), paratope of anti-N-glycolylneuraminic acid antibody (breast cancer, melanoma cancer), PLAP-like testicular alkaline phosphatase (ovarian cancer, testicular cancer), PSMA (prostate tumors), PSA (prostate), ROB04, TAG72 (tumor-associated glycoprotein 72, AML, gastric cancer, colorectal cancer, ovarian cancer), T-cell transmembrane protein (cancer), Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, carcinoma), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis-inducing ligand receptor 1, lymphoma, NHL, colorectal cancer, lung cancer), VCAM-1 (CD106, melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor-associated antigen targets have been reviewed (Gerber et al., mAbs 2009 1:247-253; Novellino et al., Cancer Immunol Immunother. 2005 54:187-207, Franke et al., Cancer Biother Radiopharm. 2000, 15:459-76, Guo et al., Adv Cancer Res. 2013; 119:421-475, Parmiani et al., J Immunol. 2007178:1975-9). Examples of these antigens include clusters of differentiation (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD4 1. CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62 E. CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90 , CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, C D138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, .CD168 , CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), Annexin A1, Nucleolin, Endoglin (CD105), ROB04, Aminopeptidase N, -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, GD2, CEA, MelanA / MART1, Ras mutant, 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, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, GD3, trehalosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYPIBI, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumin, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.

[0212] In some embodiments, the targeting moiety capable of targeting cancer is not an antibody, but is another type of targeting moiety. Table 7 (shown in Section IV below) presents some representative non-antibody targeting moieties and their corresponding targets.

[0213] In another embodiment, the targeting moiety can be a binding partner for a protein known to be expressed on cancer cells. Such expression levels can include overexpression. For example, the binding partners described in Table 8 (shown in Section IV below) can bind to the following targets on cancer cells.

[0214] The binding partner need not comprise the full length or wild type sequence 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 and thus may include truncated forms, analogs, variants and derivatives well known in the art.

[0215] Additionally, in some embodiments, the binding partner may be an aptamer that is capable of binding to a protein known to be expressed on cancer cells. Aptamers that bind to cancer cells are well known, and methods for designing them are known.

[0216] The cell-based SELEX system can be used to select a group of target cell-specific aptamers from a random candidate library. The ssDNA or ssRNA confluence can be dissolved in a binding buffer and denatured, then incubated with target cells. After washing, the bound DNA or RNA can be eluted by heating, then incubated (if necessary) with negative cells, centrifuged, and the supernatant removed. The supernatant can be amplified by PCR using a biotinylated primer. Streptavidin-coated beads can be used to separate the selected sense ssDNA or ssRNA from the antisense biotinylated chain. To increase affinity, washing intensity can be increased by increasing wash time, buffer volume, and number of washes. After the desired number of rounds, the selected ssDNA or ssRNA confluence can be PCR amplified and cloned into Escherichia coli (E. coli) and sequenced. See Shangguan et al., Aptamers evolved from live cells as effective molecular probes for cancer study, PNAS 103(32:11838-11843 (2006); Lyu et al., Generating Cell Targeting Aptamers for Nanotherapeutics Using Cell-SELEX, Theranostics 6(9):1440-1452 (2016); also see Li et al., Inhibition of Cell Proliferation by an Anti-EGFR Aptamer, PLoS One 6(6):e20229 (2011). The specific methods for designing aptamers and the specific aptamers that bind to cancer cells in these references are hereby incorporated by reference into this article.

[0217] For example, the aptamer may comprise SEQ ID NOs: 94 to 164. In some embodiments, the aptamer may comprise SEQ ID NO: 95. These aptamers are directed against EGFR and are provided merely as a representative of aptamers that can bind to targets presented on cancer cells. Other aptamers directed against other targets on cancer cells are also part of the description herein and are incorporated herein by reference, as described in Zhu et al., Theranostics 4(9):931-944 (2014).

[0218] In some embodiments, aptamers for use herein are synthesized with a K in the nanomolar to picomolar range, such as 1 picomolar to 500 nanomolar or 1 picomolar to 100 nanomolar. d Binds to its target on cancer cells.

[0219] 2. Targeting moieties that can target cells in the tumor microenvironment

[0220] Current cancer immunotherapy antibodies require effector cells to be present in the tumor. However, tumors can produce an immunosuppressive environment that excludes effector cells, resulting in less effective immunotherapy (see Herbst et al. Nature. 515(7528):563-7(2014)). Tumors lacking effector cells can be referred to as "cold tumors" (see Whiteside TL et al. Clin Cancer Res. 22(8):1845-55(2016)).

[0221] Effector cells can be excluded from tumors by the tumor stroma, in particular by non-cancerous cells in the tumor microenvironment, such as tumor-associated fibroblasts (TAFs) (see Ziani et al., Front Immunol. 9:414 (2018)). Non-cancerous cells in the tumor microenvironment can be referred to as "tumor microenvironment cells". Because T cells in cold tumors can be restricted to the stroma, immunotherapeutics targeted to the interface of malignant cells and the stroma can potentially overcome immune exclusion. Therefore, targeting tumor microenvironment cells with a second targeting moiety can provide additional specificity to the kits or compositions of the present invention.

[0222] In some embodiments, the first targeting moiety binds to an antigen antigen expressed by cancer, and the second targeting moiety binds to an antigen expressed by tumor microenvironment cells. In some embodiments, the tumor microenvironment cells are fibroblasts or macrophages. These cells may be referred to as tumor-associated fibroblasts or cancer-associated fibroblasts (TAFs or CAFs) or tumor-associated macrophages or cancer-associated macrophages (TAMs or CAMs). As used herein, "tumor-associated fibroblasts" refers to fibroblasts that are closely adjacent to a tumor mass or seen in a tumor mass. As used herein, "tumor-associated macrophages" refers to macrophages that are closely adjacent to a tumor mass or seen together with a tumor mass.

[0223] Figure 2 One component of TWICE is shown to target cancer cells, and the other component is targeted to non-cancerous cells in the tumor microenvironment. In the case of this type of TWICE, the immunological binding domains of the two components and / or the complementary binding domains of the two components will pair only when cancer cells and non-cancerous tumor microenvironment cells are in close proximity.

[0224] TAFs are characterized by the expression of activation markers such as fibroblast activation protein α (FAP) and α-smooth muscle actin (see Barnett and Vilar, J Natl Cancer Inst 110(1): 11-13 (2018)), and can create an immunosuppressive environment through direct or indirect mechanisms. Direct immunosuppression can be mediated by growth factors such as TGF-β or cytokines such as CXCL12, and indirect immunosuppression can be mediated by extracellular matrix remodeling in the tumor microenvironment (see Ziani et al., 2018).

[0225] In some embodiments, the targeting moiety of the second component binds to an antigen expressed by TAF. In some embodiments, the targeting moiety that targets TAF comprises an antibody that binds to FAP, such as sirolimus (see US20120258119A1). FAP is primarily expressed on tumor stroma but not on normal fibroblasts (Brennen et al. Mol Cancer Ther. 11(2):257–266 (2012)).

[0226] In some embodiments, the targeting moiety of the second component binds to an antigen expressed by TAM. In some embodiments, the targeting moiety targeted to TAM comprises an antibody that binds MAC-1 / CD11b (see EP0488061A2) or sideroflexin 3 (see WO2018020000).

[0227] D. Immune cell binding domain

[0228] The first immune cell binding domain and the second immune cell binding domain are capable of binding to an antigen on an immune cell when bound to each other. As used herein, an "immune cell" can be any cell included in the immune system. An immune cell can be a cell involved in an innate or adaptive immune response.

[0229] In some embodiments, the immune cell is a natural killer cell (NK), a macrophage, or a T cell. In some embodiments, the T cell is a γδ T cell or a natural killer T cell (NKT cell).

[0230] 1. T cell binding domain

[0231] In some embodiments, the first T cell binding domain and the second T cell binding domain are capable of binding to a T cell when bound to each other. In some embodiments, the first T cell binding domain and the second T cell binding domain are capable of binding to CD3 or a T cell receptor (TCR) when bound to each other.

[0232] CD3 is present on all T cells and is composed of subunits designated as γ, δ, ε, ζ and η. TCR is another molecule composed of different subunits designated as α, β, γ and δ present on T cells. The cytoplasmic tail of CD3 is sufficient to transduce the necessary signals for T cell activation in the absence of other components of the TCR receptor complex. Generally, the activation of T cell cytotoxicity depends first on the binding of TCR to the major histocompatibility complex (MHC) protein located on a separate cell, which itself binds to foreign antigens. In the absence of external intervention, only when this initial TCR-MHC binding occurs can a CD3-dependent signal cascade be achieved, which leads to T cell clonal expansion and, ultimately, the generation of T cell cytotoxicity. However, in some embodiments of the present invention, when the first T cell binding domain and the second T cell binding domain bind CD3 and / or TCR, cytotoxic T cell activation in the absence of independent TCR-MHC can occur by means of cross-linking of CD3 and / or TCR molecules, thereby simulating immune synapse formation. This means that T cells can be activated for cytotoxicity in a clone-independent manner, that is, in a manner that is independent of the specific TCR clone carried by the T cell. This allows the activation of the entire T cell compartment, rather than just specific T cells with a certain clonal identity.

[0233] In some embodiments, the first T cell binding domain is a VH domain and the second T cell binding domain is a VL domain. In some embodiments, the first T cell binding domain is a VL domain and the second T cell binding domain is a VH domain. In some embodiments, the first T cell binding domain and the second T cell binding domain, when paired together, may constitute an Fv. As used herein, "Fv" refers to a VH and a VL that are combined together. In other words, when paired together, the first T cell binding domain and the second T cell binding domain may constitute an "scFv," with the exception of the fact that the VH and VL are not in a single-chain configuration with a linker between the VH and VL.

[0234] If the first T cell binding domain and the second T cell binding domain are a pair of VH domains and VL domains, the VH domains and VL domains can be specific for an antigen expressed on the surface of a T cell, such as CD3 or TCR. In some embodiments, the anti-CD3 or anti-TCR antibody binds to a subunit of CD3 or TCR. If the antigen is CD3, a potential T cell binding domain can be derived from moromona (moromona-CD3 or OKT3), otelizumab, telizumab, visilizumab, foralizumab, SP34, or blinatumomab. Those skilled in the art will be aware of a wide range of anti-CD3 antibodies, some of which are approved therapies or have been clinically tested in human patients (see Kuhn and Weiner Immunotherapy 8(8):889-906(2016)). Table 9 (shown in Section IV below) presents selected publications on exemplary anti-CD3 antibodies. Antibodies specific for TCRs including αβ and γδ TCRs are also well known. Table 10 (shown in Section IV below) presents selected publications regarding exemplary anti-TCR antibodies.

[0235] The T cell binding domain may also include any of the antibodies listed as potential complementary binding domains, such as programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS).

[0236] 2. Natural Killer Cell Binding Domain

[0237] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of activating natural killer (NK) cells when paired together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding CD16A when paired together. In some embodiments, the binding of the paired immune cell binding domain to CD16A on NK cells activates NK cells against CD33-positive leukemia cells. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or part of the VH and / or VL of NTM-1633 (see NCT03603665) or AFM13 (see NCT01221571).

[0238] 3. Macrophage Binding Domain

[0239] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to a macrophage when paired together. In some embodiments, the macrophage is an activated macrophage.

[0240] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CSF1R when paired together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of ematuruzumab / RG7155 (NCT01494688) or IMC-CS4 (NCT01346358).

[0241] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD40 when paired together. In some embodiments, the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of CP-870,893.

[0242] E. Complementary Binding Domains

[0243] In some embodiments, the first component and the second component comprise complementary binding domains. When the complementary binding domains of the two components are paired, this results in additional functionality. This additional functionality can, for example, induce cancer cell death or increase anti-tumor immune responses. Additional functionality can also be used to make the tumor microenvironment more hospitable to immune cells.

[0244] Because both components of TWICE are targeted to cancer or the tumor microenvironment, the additional functions mediated by the pairing of complementary binding domains will occur in the tumor or its microenvironment. In some embodiments, the additional functions mediated by the paired complementary binding domains are limited to the tumor and its microenvironment, thereby reducing off-target effects. For example, this ability to restrict activity to the tumor and its microenvironment can be beneficial for activating or inhibiting target molecules with widespread expression and / or a range of physiological functions, such as TGF-β.

[0245] In some embodiments, the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to the same antigen. For example, the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to CD3 on T cells. Binding of both the paired complementary binding domains and the paired immune cell binding domains to CD3 can result in a more robust activation of an anti-cancer immune response.

[0246] In some embodiments, the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to different antigens on the same cell. For example, the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to different antigens on T cells. Binding of both the paired complementary binding domains and the paired immune cell binding domains to two different antigens on T cells can result in robust activation of an anti-cancer immune response.

[0247] In some embodiments, the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to different cells. For example, the first complementary binding domain and the second complementary binding domain, when bound to each other, can bind to cancer cells to cause cell death, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, can bind to T cells to mediate an anti-cancer immune response. Activation of cell death pathways in tumor cells and activation of an anti-cancer immune response can increase tumor regression.

[0248] In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when bound to each other, can modulate T cell, macrophage, or NK cell activity. In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when bound to each other, can bind to a co-stimulatory antigen or a co-inhibitory antigen on a T cell.

[0249] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of having cell death-inducing activity against cancer when bound to each other. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to a cell death-inducing antigen when bound to each other.

[0250] In some embodiments, the first supplementary binding domain is a VH domain and the second supplementary binding domain is a VL domain. In some embodiments, the first supplementary binding domain is a VL domain and the second supplementary binding domain is a VH domain. In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when paired together, may constitute an Fv. In other words, when paired together, the first supplementary binding domain and the second supplementary binding domain may constitute an "scFv," except for the fact that the VH and VL are not in a single-chain configuration.

[0251] In some embodiments, the VH domain of a known antibody may serve as the first supplementary binding domain, while the VL domain may serve as the second supplementary binding domain.

[0252] In some embodiments, the VL domain of a known antibody may serve as the first supplementary binding domain, while the VH domain may serve as the second supplementary binding domain.

[0253] 1. Complementary binding domains that regulate T cell activity

[0254] In some embodiments, the first complementary binding domain and the second complementary binding domain are capable of modulating TCR activity when bound to each other. In some embodiments, the first complementary binding domain and the second complementary binding domain are capable of binding to a T cell co-signaling molecule when bound to each other. T cell co-signaling molecules have been well characterized in the literature (see Chen L and Flies DB Nat Rev Immunol 13(4):227–242 (2013)) and include co-stimulatory and co-inhibitory antigens. In some embodiments, the co-signaling molecule is a member of the immunoglobulin superfamily (IgSF) or the tumor necrosis factor receptor superfamily (TNFRSF). Co-inhibitory antigens may also be referred to as immune checkpoint molecules.

[0255] Table 11 (shown in Section IV below) provides examples of clinical investigational antibodies that may induce anti-tumor responses by modulating T cell function.

[0256] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD3 epsilon when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of muromonabin, otelizumab, telizumab, vesilizumab, foralizumab, SP34, or blinatumomab.

[0257] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to programmed cell death protein 1 (PD-1) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of pembrolizumab or nivolumab.

[0258] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of ipilimumab.

[0259] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to T cell immunoglobulin and mucin domain 3 (TIM-3) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of TSR-022 or Sym023.

[0260] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to lymphocyte activation gene 3 (LAG-3) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of BMS-986016.

[0261] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to a killer cell immunoglobulin-like receptor (KIR) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of lirelizumab.

[0262] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD28 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of cilalizumab.

[0263] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD 137 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of umelumab or usulelumab.

[0264] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to OX40 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of PF-04518600 or BMS986178.

[0265] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD27 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of varizumab.

[0266] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to GITR when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of GWN323 or BMS-986156.

[0267] In some embodiments, the first supplementary binding domain and the second supplementary binding domain, when paired together, are capable of binding to a T-cell immunoreceptor having Ig and ITIM domains (TIGIT). In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of OMP-313M32, MTIG7192A, BMS-986207, or MK-7684.

[0268] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to inducible T cell co-stimulator (ICOS) when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of JTX-2011, an anti-ICOS antibody in clinical development.

[0269] 2. Complementary binding domains targeting tumor-associated macrophages (TAMs)

[0270] In some embodiments, binding of the first and second complementary binding domains to antigens on TAMs mediates an increased immune response to the tumor. For example, anti-CSF1R blocking antibodies reduce TAM infiltration and promote CD8+ T cell expansion (Ries CH et al. Cancer Cell. 25(6):846-59 (2014)).

[0271] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of inhibiting TAMs when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of reprogramming or recruiting activated macrophages when paired together.

[0272] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CSF1R when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of ematuruzumab / RG7155 (NCT01494688) or IMC-CS4 (NCT01346358).

[0273] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD40 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of CP-870,893.

[0274] 3. Complementary binding domains for NK cell stimulation

[0275] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of activating NK cells when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to CD16A when paired together. In some embodiments, binding of the paired supplementary binding domain to CD16A on NK cells activates NK cells against CD33-positive leukemia cells. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or part of the VH and / or VL of NTM-1633 (see NCT03603665) or AFM13 (see NCT01221571).

[0276] 4. Inhibit complementary binding domains of checkpoint molecules expressed by cancer cells

[0277] In some embodiments, the first complementary binding domain and the second complementary binding domain can suppress the check point molecules expressed by cancer cells when paired together. In some embodiments, the first complementary binding domain and the second complementary binding domain can block the signal of inhibition immune cell activation from cancer cells when paired together. Many different immune check point molecules have been described in the literature (see Park et al., Experimental & Molecular Medicine 50: 109 (2018), the document is incorporated herein by reference in its entirety to disclose different immune check point molecules).

[0278] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding to PD-L1 when paired together. In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of atezolizumab, durvalumab, or avelumab.

[0279] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding CD73 when paired together.

[0280] In some embodiments, the first supplemental binding domain and the second supplemental binding domain comprise all or a portion of the VH and / or VL of CPI-006 or MEDI9447.

[0281] 5. Complementary binding domains that bind to RANK

[0282] In some embodiments, the first complementary binding domain and the second complementary binding domain are capable of inhibiting activation of regulatory T cells when paired together.

[0283] In some embodiments, the complementary binding domains bind to RANK or RANKL when paired together. RANK is expressed on cancer cells and can bind to RANKL on regulatory T cells to create an immunosuppressive environment (see de Groot et al., Cancer Treatment Reviews 62: 18-28 (2018)). In some embodiments, binding to RANK or RANKL on cancer cells blocks regulatory T cell activation and stimulates an immune response.

[0284] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding RANK when paired together.

[0285] In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of binding RANKL when paired together.

[0286] In some embodiments, the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of denosumab.

[0287] 6. Complementary binding domains that bind cell death-inducing antigens

[0288] As used herein, a "cell death-inducing" antigen is an antigen that stimulates cell death after being bound by a ligand. All types of cell death are included. Cell death can be programmed (e.g., apoptosis) or unprogrammed (e.g., necrosis). In some embodiments, cell death is necrosis, apoptosis, or programmed necrosis.

[0289] In some embodiments, the cell death-inducing antigen comprises a death receptor. Death receptors are members of the tumor necrosis factor receptor superfamily and are characterized by the presence of an intracellular "death domain." In some embodiments, the death receptor is Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER, DR6, or CAR1.

[0290] In some embodiments, the first and second complementary binding domains are capable of binding to TRAIL-R1 / DR4 when paired together. In some embodiments, the first and second complementary binding domains comprise all or a portion of the VH and / or VL of mapatumumab (an anti-TRAIL-R1 / DR4 death receptor antibody). In one embodiment, the anti-DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER antibody comprises canatumumab (AMG655), lexalimumab, tegtuzumab (CS1008), or zozituzumab (PRO95780).

[0291] F. Dimerization domain

[0292] In some embodiments, the first component and the second component further comprise a dimerization domain, such as Figure 6 As used herein, a "dimerization domain" refers to an amino acid sequence that allows two protein monomers (i.e., a single protein) to bind together. Thus, a dimerization domain can comprise any sequence that causes two protein monomers to form a dimer. In some embodiments, the binding is covalent. In some embodiments, the binding is non-covalent.

[0293] The dimerization domain can be linked to a complementary binding domain or an immune cell binding domain.

[0294] In some embodiments, the dimerization domain promotes the association of the first supplementary binding domain with the first immune cell binding domain and / or the association of the second supplementary binding domain with the second immune cell binding domain. In this way, dimerization can reduce the potential for the supplementary binding domain to dissociate from the immune cell binding domain away from the tumor site.

[0295] In some embodiments, a cleavable linker (i.e., a linker comprising a cleavage site) mediates the dissociation of the first immune cell binding domain from the first complementary binding domain of the first component and / or the dissociation of the second immune cell binding domain from the second complementary binding domain. In some embodiments, the first immune cell binding domain binds to the first complementary binding domain until at least one cleavable linker is cleaved, such that either (or both) of the first immune cell binding domain and the first complementary binding domain are released from their corresponding dimerization domain. In some embodiments, the second immune cell binding domain binds to the second complementary binding domain until at least one cleavable linker is cleaved, such that either (or both) of the second immune cell binding domain and the second complementary binding domain are released from their corresponding dimerization domain.

[0296] In some embodiments, the first immune cell binding domain is bound to the first complementary binding domain through a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the first immune cell binding domain through a first linker; the second dimerization domain is connected to the first complementary binding domain through a second linker; and the first linker and / or the second linker is a cleavable linker.

[0297] In some embodiments, the second immune cell binding domain is bound to the second complementary binding domain through a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the second immune cell binding domain through a first linker; the second dimerization domain is connected to the second complementary binding domain through a second linker; and the first linker and / or the second linker is a cleavable linker.

[0298] The dimerization domain may comprise all or a portion of a known dimerization domain amino acid sequence. The dimerization domain may comprise a sequence that has homology to a known dimerization amino acid sequence but does not match the known sequence. In some embodiments, the dimerization domain may comprise amino acids that are optimized to improve binding.

[0299] In some embodiments, the dimerization domain comprises an amino acid sequence from a transcription factor or a receptor / ligand pair. Non-limiting examples of dimerization domains include sequences from receptor tyrosine kinases, transcription factors, 14-3-3 proteins, and G protein-coupled receptors.

[0300] In some embodiments, the dimerization domain comprises a leucine zipper (also known as a coiled coil). As used herein, a "leucine zipper" refers to any amino acid sequence comprising a periodic repeat of leucine residues. A wide variety of leucine zippers for dimerization have been described, such as in US Pat. No. 9,994,646.

[0301] In some embodiments, the dimerization domain comprises an immunoglobulin domain.

[0302] In some embodiments, the dimerization domain comprises an immunoglobulin variable domain. In some embodiments, the immunoglobulin variable domain is a VH or VL domain.

[0303] In some embodiments, the dimerization domain comprises an immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain is an Fc domain. In some embodiments, the immunoglobulin constant domain is CH1 / CL, CH2, CH3, or CH4.

[0304] In some embodiments, the dimerization domain comprises an IgE CH2 domain.

[0305] In some embodiments, the dimerization domain comprises a TCR constant domain.The TCR constant domain can be from any chain (e.g., α, β, γ, or δ).

[0306] In some embodiments, the dimerization domain in the first component is the same as the dimerization domain in the second component. For example, the dimerization domains in the first component and the second component can be leucine zippers. When the dimerization domains in the first component and the second component are the same, this can be referred to as "homodimerization."

[0307] In some embodiments, the dimerization domain in the first component is different from the dimerization domain in the second component. For example, the dimerization domains in the first component and the second component can comprise a receptor / ligand pair. When the dimerization domains in the first component and the second component are different, this can be referred to as "heterodimerization."

[0308] Dimerization domains can also be engineered to create a "knob" or "hole" to dimerize two proteins. Such knob-into-hole dimerization sequences can include knob-into-hole Fc or knob-into-hole C. H A wide range of knob-in-hole examples have been described for generating bispecific antibodies (see Xu et al., mAbs 7(1):231-242 (2015)).

[0309] In some embodiments, the dimerization domain is engineered. By "engineered," it is meant that the amino acid sequence is mutated to alter one or more properties of the protein. In some embodiments, when two protein domains would not normally associate in the absence of engineering, engineering makes the association of the two domains electrostatically favorable. In some embodiments, two immunoglobulin constant domains are engineered and may be referred to as "engineered immunoglobulin constant domains." In some embodiments, engineering of the Fc region results in heterodimerization of the Fc region, such as the CH domain.

[0310] In some embodiments, the two Fc regions are engineered to contain opposite charges and associate together (see WO2009089004). In some embodiments, the two Fc regions are engineered to contain two leucine zippers and associate together (see US9,994,646). In some embodiments, the two Fc regions are engineered by lysine repositioning (see WO2017106462).

[0311] The domains can be engineered so that each component of TWICE is sufficiently stable during manufacturing, transport, and administration, but the binding kinetics will still favor pairing of the immunobinding domains with each other, and, when applicable, of the complementary functional domains with each other, when the two components of TWICE are in close proximity.

[0312] Several ways to generate heterodimeric Fc can be used to prepare the constructs described herein. In an attempt to generate bispecific antibodies in the form of asymmetric IgG, i.e., IgG formed as a heterodimer by two different paired heavy chains, Ridgway et al. have engineered the CH3 domain of IgG1 by mutation so that one chain contains a "knob" and the other chain contains a corresponding "hole" (JB Ridgway et al. Protein Eng. 9: 617–621 (1196)). Due to steric hindrance in homodimers, the two engineered heavy chains of this "knob-in-hole" approach will preferentially form heterodimers when co-expressed from the same cell. Since then, several other mutations have been disclosed that use similar steric hindrance or repulsive charge strategies in engineered Fc domains to promote heterodimer formation between two different chains (Atwell et al. Journal of Molecular Biology 270: 26-35 (1997); Gunasekaran et al., Journal of Biological Chemistry 285: 19637-46 (2010); Moore et al. mAbs 3, 546–557 (2011); Strop P et al. J. Mol. Biol. 420, 204–219 (2012), Von Kreudenstein et al. 2013, mAbs; 5: 646-54 (2013)). A combination of steric hindrance and repulsive charge has also been used to engineer IgG heavy chains to efficiently form heterodimers (WO2017106462A1). Using a different approach, Davis et al. (Davis et al., Protein Engineering, Design & Selection 23(4):195–202 (2010)) have developed strand exchange engineered domains (SEEDs) using IgG and IgA sequences to engineer asymmetric CH3 domains that enable Fc heterodimerization.

[0313] G. Supplementary functional domains

[0314] In some embodiments, the first component or the second component comprises a complementary functional domain. As used herein, a "complementary functional domain" refers to a domain that has a function when bound to a specific cell type. A complementary functional domain is different from a complementary binding domain because a complementary functional domain does not need to be paired with another domain to have a function.

[0315] In some embodiments, both the first component and the second component comprise a supplementary functional domain. In some embodiments, only the first component comprises a supplementary functional domain.

[0316] In general, the extracellular domain of a cell surface protein that acts as a ligand for a receptor can be used as a supplementary functional domain. In some embodiments, the supplementary functional domain is a co-stimulatory molecule for T cell activation. In some embodiments, the supplementary functional domain is a ligand for binding to an integrin.

[0317] In some embodiments, the supplementary functional domain is a latent form of a member of the TGF-β family. In some embodiments, the latent form is activated in the tumor microenvironment to act locally at the tumor cell.

[0318] In some embodiments, the supplementary functional domain is a cytokine. A wide range of cytokines have been clinically tested in the case of cancer (see Vazquez-Lombardi et al., Antibodies 2: 426-451 (2013)). In addition, the incorporation of cytokines as the supplementary functional domain of TWICE allows local delivery of cytokines, and therefore, the systemic administration of cytokines can be improved. In some embodiments, the cytokine is a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ or TNF superfamily.

[0319] In some embodiments, the supplemental functional domain is an attenuated cytokine.

[0320] As used herein, an "attenuated cytokine" is a cytokine that has a mutation that reduces its activity compared to a wild-type cytokine, or a cytokine that is "masked" with a mask that is attached to the cytokine via a linker that can be cleaved in the tumor or tumor matrix microenvironment. Thus, an attenuated cytokine can be a variant form of a naturally occurring cytokine. In some embodiments, the attenuated cytokine lacks activity unless it is targeted to the target cell. Attenuated cytokines targeted to tumors can have robust anti-tumor effects with limited systemic toxicity (see Pogue et al., PLoS ONE 11(9):e0162472 (2016) and Pogue et al., Cytokine 1:66 (2015)).

[0321] It has also been proposed that attenuated cytokines activate the immune system and induce the proliferation of effector T cells, whereas the same cytokines that are not attenuated may lead to suppression of the immune system and the proliferation of regulatory T cells.

[0322] In some embodiments, the attenuated cytokine is a variant form of IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or a member of the TNF superfamily. In some embodiments, the attenuated cytokine must be targeted to the cancer or its microenvironment to be functional.

[0323] H.Connector

[0324] By linker we include any chemical moiety that joins the parts of TWICE together.

[0325] In some embodiments, the linker used in TWICE can be a flexible linker. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic substances (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. The length of the peptide linker can be about 2-100, 10-50, or 15-30 amino acids. In some embodiments, the length of the peptide linker can be at least 10, at least 15, or at least 20 amino acids, and at most 80, at most 90, or at most 100 amino acids. In some embodiments, the linker is a peptide linker having a single or repeating 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.

[0326] In some embodiments, the linker does not comprise a cleavage site (ie, a non-cleavable linker). Exemplary linkers that do not comprise a cleavage site include maleimide (MPA) or SMCC linkers.

[0327] In some embodiments, the linker comprises a cleavage site (ie, a cleavable linker).

[0328] In some embodiments, the linker connects the targeting moiety to the immune cell binding domain, the complementary binding domain, or the complementary functional domain. In some embodiments, the linker connects the dimerization domain to the immune cell binding domain or the complementary binding domain.

[0329] 1. Linkers for connecting targeting moieties

[0330] In some embodiments, the linker connects the targeting moiety to the immune cell binding domain. In some embodiments, the linker connects the targeting moiety to the complementary binding domain or complementary functional domain. In some embodiments, the linker connecting the targeting moiety to the complementary binding domain or complementary functional domain is a non-cleavable linker. In some embodiments, the linker connecting the targeting moiety to the complementary binding domain or complementary functional domain is a flexible linker.

[0331] 2. Linkers for connecting dimerization domains

[0332] In some embodiments, a linker connects the dimerization domain to the immune cell binding domain. In some embodiments, a linker connects the dimerization domain to the complementary binding domain.

[0333] A linker used to connect the dimerization domain to the immune cell binding domain or the complementary binding domain may be referred to as a “dimerization domain linker.” In some embodiments, the dimerization domain linker is a cleavable linker.

[0334] In some embodiments, the dimerization domain linker may comprise a cleavage site. In some embodiments, the dimerization domain linker comprises a protease cleavage site. In some embodiments, the dimerization domain linker comprises a protease cleavage site that can be cleaved in the tumor microenvironment. In some embodiments, the dimerization domain linker comprises a cleavage site for one or more matrix metalloproteinases.

[0335] Depending on the dimerization domain employed, a range of dimerization domain linker lengths can be used in TWICE. The length of the dimerization domain linker may need to be long enough to allow for flexibility in protease cleavage. However, a dimerization domain linker that is too long may cause the two dimerization domains to be too far apart in space to ensure dimerization.

[0336] In some embodiments, the first dimerization domain linker and the second dimerization domain linker have the same or similar length.In some embodiments, the first dimerization domain linker and the second dimerization domain linker have different lengths.

[0337] In some embodiments, the dimerization domain linker length helps promote the association of the two dimerization domains.

[0338] In some embodiments, the first dimerization domain linker and the second dimerization domain linker are 5-30 amino acids in length. In some embodiments, the first dimerization domain linker and the second dimerization domain linker are 8-16 amino acids in length.

[0339] In some embodiments, the first dimerization domain linker and the second dimerization domain linker are 5-15 amino acids in length. For example, when the dimerization domain is CH1 / CL, a linker of this length may be appropriate.

[0340] In some embodiments, the first dimerization domain linker and the second dimerization domain linker are 12-30 amino acids in length. For example, when the dimerization domain is a leucine zipper (coiled coil), a linker of this length may be appropriate. In some embodiments, the first dimerization domain linker and the second dimerization domain linker are 15-20 amino acids in length. For example, a linker of this length may be suitable for certain leucine zipper dimerization domains.

[0341] Examples of representative dimerization domain linkers include SEQ ID NOs: 203-211.

[0342] I. Cleavage Sites and Cleavable Linkers

[0343] In some embodiments, the cleavage site allows for specific cleavage of the construct at certain positions.Linkers comprising a cleavage site may be referred to as cleavable linkers.

[0344] For example, a TWICE that is a single polypeptide construct may include a cleavable linker between the first component and the second component. Other embodiments of this TWICE may also employ a cleavable linker, such as a dimerization domain linker.

[0345] In some embodiments, cleavage can occur outside of the undesired cell without first being internalized into the cell and engaging in the classical antigen processing pathway.

[0346] In certain embodiments, at least one cleavage site is cleavable by an enzyme expressed by the cancer cell. For example, cancer cells are known to express certain enzymes, such as proteases. By way of non-limiting example, cathepsin B cleaves FR, FK, VA, and VR, among other sequences; cathepsin D cleaves PRSFFRLGK (SEQ ID NO: 45); ADAM28 cleaves KPAKFFRL (SEQ ID NO: 1), DPAKFFRL (SEQ ID NO: 2), KPMKFFRL (SEQ ID NO: 3), and LPAKFFRL (SEQ ID NO: 4); and MMP2 cleaves, for example, AIPVSLR (SEQ ID NO: 46), SLPLGLWAPNFN (SEQ ID NO: 47), HPVGLLAR (SEQ ID NO: 48), GPLGVRGK (SEQ ID NO: 49), and GPLGLWAQ (SEQ ID NO: 50). Other cleavage sites listed in Table 1 or Table 2 may also be used. Protease cleavage sites and proteases associated with cancer are well known in the art. Oncomine (www.oncomine.org) is an online cancer gene expression database, so when the agent of TWICE is used to treat cancer, the skilled person can search the Oncomine database to identify the specific protease cleavage site (or two protease cleavage sites) that will be suitable for treating a given cancer type. Alternative databases include the European Bioinformatic Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk / gxa). Protease databases include ExPASy Peptide Cutter (www.ca.expasy.org / tools / peptidecutter).

[0347] In some embodiments, the protease is expressed by non-cancerous cells in the tumor microenvironment, such as tumor-associated macrophages or fibroblasts.

[0348] In some embodiments, the protease cleavage site of one or more cleavable linkers is cleaved by a co-localized protease. In some embodiments, the protease is co-localized to the cancer via a targeting moiety that binds to a tumor antigen expressed by the cancer and is the same or different from the targeting moiety in the first component or the second component. In some embodiments, the protease is co-localized to the cancer via a targeting moiety that binds to an antigen expressed by cells in the tumor microenvironment.

[0349] The cleavage site in the cleavable linker can serve to release the dimerization domain from the complementary binding domain and / or the immune cell binding domain. The cleavage site can serve to release the complementary binding domain and / or the immune cell binding domain from the first and / or immune cell engaging domain in the microenvironment of an undesirable cell.

[0350] In some embodiments, the protease cleavage site in the first cleavable linker and / or the second cleavable linker connecting the dimerization domain to the immune cell binding domain or the complementary binding domain is cleaved by a protease expressed by the cancer or is co-localized to the cancer by a targeting moiety that binds to a tumor antigen expressed by the cancer.

[0351] J. Binding of the Complementary Binding Domain to the Immune Cell Binding Domain

[0352] In some embodiments, the first supplementary binding domain is a binding partner of 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 first immune cell binding domain does not bind to the first supplementary domain.

[0353] In some embodiments, the second supplementary binding domain is a binding partner of 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 second supplementary domain does not bind to the second supplementary domain.

[0354] When a complementary binding domain dissociates from an immune cell binding domain and associates with a paired complementary domain, or when an immune cell binding domain dissociates from a complementary binding domain and associates with a paired immune cell binding domain, this can be referred to as domain swapping. In this domain swapping event, the domains shift from an unmatched configuration to an active and paired configuration.

[0355] In some embodiments, the first immune cell binding domain and the second immune cell binding domain are capable of forming an Fv when not bound to the first supplementary binding domain and the second supplementary binding domain. In some embodiments, the first supplementary binding domain and the second supplementary binding domain are capable of forming an Fv when not bound to the first immune cell binding domain and the second immune cell binding domain.

[0356] In some embodiments, the first component and the second component comprise a dimerization domain to promote the association of the immune cell binding domain and the complementary binding domain of the single component. In some embodiments, cleavage of a cleavable linker in the dimerization domain linker allows the dissociation of the immune cell binding domain and the complementary binding domain of the single component to be achieved at the cancer cell or its microenvironment. In some embodiments, the first component and the second component do not comprise a dimerization domain.

[0357] K. Masking by Inert Binding Partners

[0358] In some embodiments, TWICE comprises an inert binding partner to mask the first immune cell binding domain and / or the second immune cell binding domain. In some embodiments, only the first immune cell binding domain is masked. In some embodiments, both the first immune cell binding domain and the second immune cell binding domain are masked.

[0359] For example, in some embodiments, the inert binding partner is a component of those TWICEs that include a supplementary functional domain, such as Figure 5 and those described in Section G.

[0360] In some embodiments, the first component comprising the complementary functional domain and / or the second component comprises an inert binding partner.

[0361] In some embodiments, the first inert binding partner 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.

[0362] In some embodiments, the first immune cell binding domain is a VH domain and the inert binding partner is a VL domain. In some embodiments, the first immune cell binding domain is a VL domain and the inert binding partner is a VH domain.

[0363] In some embodiments, the protease cleavage site separates the first immune cell binding domain and the first inert binding partner. In some embodiments, the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease.

[0364] In some embodiments, 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.

[0365] In some embodiments, the second immune cell binding domain is a VH domain and the inert binding partner is a VL domain. In some embodiments, the second immune cell binding domain is a VL domain and the inert binding partner is a VH domain.

[0366] In some embodiments, the protease cleavage site separates the second immune cell binding domain and the second inert binding partner. In some embodiments, the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease.

[0367] In some embodiments, the protease is expressed by a cancer. In some embodiments, the protease is expressed by cells in the tumor microenvironment, such as TAFs or TAMs.

[0368] In some embodiments, the protease is co-localized to the cancer by a targeting moiety that is an antibody or antigen-binding fragment that binds (a) a tumor antigen expressed by the cancer and is the same as or different from the first targeting moiety and / or the second targeting moiety in the agent, or (b) binds an antigen expressed by cells in the tumor microenvironment.

[0369] L. Preparation method

[0370] The various TWICEs described herein can be prepared using genetic engineering techniques. Specifically, one or both nucleic acids can be expressed in a suitable host to produce a TWICE or its components. For example, a vector can be prepared containing a nucleic acid sequence encoding a TWICE, including all of its components and linkers, and that vector can be used to transform an appropriate host cell (or a single vector can be used to express each component separately).

[0371] Various regulatory elements may also be employed in the vector, depending on the nature of the host and the manner in which the nucleic acid is introduced into the host, and whether episomal maintenance or integration is desired.

[0372] Chemical ligation techniques, such as the use of maleimide or SMCC linkers, may also be employed.

[0373] In the case where the targeting moiety is an aptamer, one of ordinary skill in the art will understand how to conjugate the aptamer to a protein, i.e., an immune cell binding domain or a complementary binding domain. Aptamers can be conjugated using thiol linkages or other standard conjugation chemistries. Maleimide, succinimide, or SH groups can be attached to the aptamer to link it to the immune cell binding domain or the complementary binding domain.

[0374] II. Pharmaceutical Compositions

[0375] TWICE can be used as a pharmaceutical composition. Therefore, they can be prepared together with a pharmaceutically acceptable carrier. If parenteral administration is desired, TWICE can be provided, for example, in sterile, pyrogen-free water for injection or sterile, pyrogen-free saline, or other forms acceptable for parenteral administration. Alternatively, TWICE can be provided in a lyophilized form for resuspending with the addition of a sterile liquid carrier. If provided separately, TWICE can be provided as a single pharmaceutical composition or as two pharmaceutical compositions.

[0376] III. How to use TWICE

[0377] The TWICE described herein can be used in a method of treating cancer comprising administering to a patient a TWICE comprising at least a first component and a second component, each of which has been described in detail in various embodiments above.

[0378] Additionally, the agents described herein can also be used in a method of targeting a patient's own immune response to cancer cells, the method comprising administering TWICE to the patient.

[0379] In some embodiments, TWICE described herein can be used in a method of targeting an immune cell to a cancer that expresses a tumor antigen that binds both a first targeting moiety and a second targeting moiety. In some embodiments, the immune cell is a T cell that expresses CD3 or a TCR.

[0380] In some embodiments, TWICE described herein can be used in a method of targeting an immune cell to a cancer that expresses two tumor antigens, wherein one tumor antigen binds a first targeting moiety and one tumor antigen binds a second targeting moiety. In some embodiments, the immune cell is a T cell that expresses CD3 or TCR.

[0381] In some embodiments, TWICE described herein can be used in methods for delivering cytokines to immune cells of a patient. In some embodiments, the first supplementary functional domain and / or the second supplementary functional domain of TWICE comprises a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

[0382] In some embodiments, the patient suffers from cancer or has a pre-malignant state identified. In some embodiments, the patient suffers from undetectable cancer, but is at high risk of developing cancer, including having a mutation associated with an increased risk of cancer. In some embodiments, the patient at high risk of developing cancer suffers from a pre-malignant tumor with a high risk of transformation. In some embodiments, the patient at high risk of developing cancer has a genetic profile associated with high risk. In some embodiments, the presence of cancer or a pre-malignant state in the patient is determined based on the presence of circulating tumor DNA (ctDNA) or circulating tumor cells. In some embodiments, treatment is preemptive or preventive. In some embodiments, treatment slows down or prevents the occurrence or recurrence of cancer.

[0383] The amount of agent administered to the patient can be selected by the patient's physician so as to provide an amount effective to treat the condition in question. The first and second components of TWICE can be administered in the same formulation, or in two different formulations administered within a time period sufficiently close to be active in the patient.

[0384] The patient to be treated can be a human. The patient can be a primate or any mammal. Alternatively, the patient can be an animal, such as a domesticated animal (e.g., a dog or cat), a laboratory animal (e.g., a laboratory rodent, such as a mouse, rat, or rabbit), or an animal important in agriculture (e.g., a horse, cow, sheep, or goat).

[0385] Cancer can be a solid or non-solid malignant tumor. In some embodiments, the cancer can be a solid tumor, wherein the solid tumor is not a lymphoma. The cancer can be any cancer, such as breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, stomach cancer, pancreatic cancer, colorectal 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 diseases, and pre-malignant diseases or any other cancer suitable for treatment.

[0386] In some embodiments, one skilled in the art can assess the tumor prior to initiating treatment.

[0387] For example, one skilled in the art can assess the presence of infiltrating T cells in a tumor. In some embodiments, the treatment method includes confirming the presence of T cells in the tumor before initiating treatment. In some embodiments, the patient will have a solid tumor with T cell infiltration. The tumor sample can be stained for T cell markers, and imaging techniques can be used to show the presence, and optionally the concentration, of T cell infiltration in the tumor.

[0388] In some embodiments, the treatment method includes confirming the presence of tumor antigens before initiating treatment. In some embodiments, the treatment method includes assessing the concentration of tumor antigens before initiating treatment. For example, one skilled in the art can select tumor antigens or select patients for treatment based on the profile of antigens overexpressed by the tumor. One skilled in the art can also select patients for treatment with TWICE based on their antigen expression profiles by selecting patients who overexpress specific antigens. In some embodiments, these tumor antigens are cell surface tumor antigens. Tumor samples can be stained with respect to the target antigen to show the presence of tumor antigens, and optionally display the concentration. The lower limit of expression can also be determined using an in vitro T cell activation assay performed with a specific TWICE. These methods allow researchers to confirm that a patient's tumor has antigens suitable for TWICE targeting to the tumor microenvironment.

[0389] In some embodiments, the treatment method comprises (i) confirming the presence of T cells in the tumor; (ii) confirming the presence of one or more tumor antigens, and / or (iii) assessing the concentration of the tumor antigens. In some embodiments, the expression of the one or more tumor antigens that bind the first targeting moiety and the second targeting moiety is expressed at a sufficiently high level to bring the two components of TWICE into close proximity.

[0390] In some embodiments, the presence of a biomarker is used to select patients to receive TWICE. A wide variety of tumor markers are known in the art, such as those described at www.cancer.gov / about-cancer / diagnosis-staging / diagnosis / tumor-markers-fact-sheet. In some embodiments, the tumor marker is 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; carcinoembryonic antigen (CEA); CD20; chromogranin A (CgA); chromosome 3, 7, 17, or 9p21; circulating tumor cells of epithelial origin Cytokeratin fragment 21-1; EGFR gene mutation analysis; estrogen receptor (ER) / progesterone receptor (PR); fibrin / fibrinogen; HE4; HER2 / neu gene amplification or protein overexpression; immunoglobulins; KRAS gene mutation analysis; 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 activator inhibitor (PAI-1); 5 protein signatures 21 gene signatures (Oncotype ); or 70 gene characteristics

[0391] TWICE can be administered alone or in combination with other forms of therapy, including surgery, radiation, traditional chemotherapy, or other immunotherapies.

[0392] In some embodiments, other immunotherapies may include separate treatments with immune cytokines or cytokine fusions. Cytokines refer to cell signaling proteins that are naturally produced by the body to activate and regulate the immune system. Cytokine fusions refer to engineered molecules that comprise all or part of a cytokine. For example, a cytokine fusion may comprise all or part of an antibody that is connected to a cytokine that allows targeting to a tumor, such as Darleukin (see Zegers et al. (2015) Clin. Cancer Res., 21, 1151-60), Teleukin (see WO2018087172).

[0393] In some embodiments, other immunotherapies are cancer treatment vaccinations. In some embodiments, cancer treatment vaccinations strengthen the body's natural defenses to fight cancer. These can be directed against shared tumor antigens (such as E6, E7, NY-ESO, MUC1, or HER2) or against personalized mutant neoantigens.

[0394] IV. Implementation Plan

[0395] The following numbered items provide embodiments as described herein, but the embodiments described herein are not limiting.

[0396] Item 1. A kit or composition for treating cancer in a patient, the kit or composition comprising a first component, the 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; a first immune cell binding domain capable of having immune cell binding activity when bound to a second immune cell binding domain, wherein the second immune cell binding domain is not part of the first component, and wherein the first immune cell binding domain is a VH domain or a VL domain; and a first complementary binding domain capable of binding to a complementary antigen when bound to a second complementary binding domain, wherein the second complementary binding domain is not part of the first component, when the first immune cell binding domain is a VH domain, the first complementary binding domain is a VL domain, when the first immune cell binding domain is a VL domain, and wherein the first complementary binding domain is a binding partner of the first immune cell binding domain, such that the first immune cell binding domain binds to the complementary antigen unless the first immune cell binding domain does not bind to the first complementary domain. wherein the second immune cell binding domain is a VL domain and the second immune cell binding domain is a VH domain; and wherein the second immune cell binding domain is a VH domain and the second immune cell binding domain is a VL domain.

[0397] Item 2. The kit or composition of Item 1, wherein the first immune cell binding domain is bound to the first complementary binding domain via a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the first immune cell binding domain via a first linker; the second dimerization domain is connected to the first complementary binding domain via a second linker; and the first linker and / or the second linker is a cleavable linker.

[0398] Item 3. The kit or composition of any one of items 1-2, wherein the second T cell binding domain is bound to the second complementary binding domain via a first dimerization domain and a second dimerization domain, wherein the first dimerization domain is connected to the second T cell binding domain via a first linker; the second dimerization domain is connected to the second complementary binding domain via a second linker; and the first linker and / or the second linker is a cleavable linker.

[0399] Item 4. The kit or composition of any one of Items 2-3, wherein the first linker and the second linker are cleavable linkers.

[0400] Item 5. The kit or composition of any one of Items 2-4, wherein the first linker and the second linker are the same.

[0401] Item 6. The kit or composition of any one of Items 2-4, wherein the first linker and the second linker are different.

[0402] Item 7. The kit or composition of any one of Items 2-6, wherein the first linker and the second linker are 5 to 30 amino acids in length.

[0403] Item 8. The kit or composition of any one of Items 2-6, wherein the first linker and the second linker are 8 to 16 amino acids in length.

[0404] Item 9. The kit or composition of any one of Items 2 to 8, wherein the protease cleavage site of the first cleavable linker and / or the second cleavable linker is cleaved by a protease expressed by cells of the cancer or tumor microenvironment.

[0405] Item 10. The kit or composition of any one of items 2 to 9, wherein the protease cleavage site of the first cleavable linker and / or the second cleavable linker is cleaved by a protease that is co-localized to the cancer by a targeting moiety that is an antibody or antigen-binding fragment thereof, which binds to a tumor antigen expressed by the cancer and is the same as or different from the targeting moiety in at least one of the first component or the second component.

[0406] Item 11. The kit or composition of any one of Items 1-10, wherein the first dimerization domain and the second dimerization domain are both leucine zippers; immunoglobulin domains; or T cell receptor (TCR) domains.

[0407] Item 12. The kit or composition of Item 11, wherein the immunoglobulin domain comprises an immunoglobulin variable domain or an immunoglobulin constant domain.

[0408] Item 13. The kit or composition of Item 12, wherein the immunoglobulin constant domain comprises CH1 / CL, CH2, CH3, or CH4.

[0409] Item 14. The kit or composition of Item 11, wherein the TCR domain comprises a TCR constant domain.

[0410] Item 15. The kit or composition of any one of items 1 to 14, wherein the dimerization domain in the first component is the same as the dimerization domain in the second component.

[0411] Item 16. The kit or composition of any one of items 1 to 14, wherein the dimerization domain in the first component is different from the dimerization domain in the second component.

[0412] Item 17. The kit or composition of any one of Items 1 to 16, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to the cancer when associated with each other.

[0413] Item 18. The kit or composition of Item 17, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to an immune checkpoint molecule, RANK or RANKL, or a cell death-inducing antigen when bound to each other.

[0414] Item 19. The kit or composition of Item 18, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to an immune checkpoint molecule when bound to each other.

[0415] Item 20. The kit or composition of Item 19, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding to PD-L1 when associated with each other.

[0416] Item 21. The kit or composition of Item 20, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of atezolizumab, durvalumab, or avelumab.

[0417] Item 22. The kit or composition of Item 19, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding CD73 when associated with each other.

[0418] Item 23. The kit or composition of Item 22, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of CPI-006 or MEDI9447.

[0419] Item 24. The kit or composition of Item 18, wherein the first complementary binding domain and the second complementary binding domain are capable of binding RANK when associated with each other.

[0420] Item 25. The kit or composition of Item 18, wherein the first complementary binding domain and the second complementary binding domain are capable of binding RANKL when associated with each other.

[0421] Item 26. The kit or composition of Item 25, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of denosumab.

[0422] Item 27. The kit or composition of Item 18, wherein the first complementary binding domain and the second complementary binding domain are capable of binding a cell death-inducing antigen when associated with each other.

[0423] Item 28. The kit or composition of Item 27, wherein the first supplementary binding domain and the second supplementary binding domain, when associated with each other, are capable of binding to Fas / CD95 / Apo1, TNFR1 / p55 / CD120a, DR3 / Apo3 / WSL-1 / TRAMP / LARD, TRAIL-R1 / DR4, DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER, DR6 or CAR1.

[0424] Item 29. The kit or composition of Item 28, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding TRAIL-R1 / DR4 when associated with each other.

[0425] Item 30. The kit or composition of Item 29, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of mapatumumab.

[0426] Item 31. The kit or composition of Item 28, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding to DR5 / Apo2 / TRAIL-R2 / TRICK2 / KILLER when associated with each other.

[0427] Item 32. The kit or composition of Item 31, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of canatumumab (AMG655), lexalimumab, tegasuizumab (CS1008), or zozituzumab (PRO95780).

[0428] Item 33. The kit or composition of any one of items 1 to 16, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to a molecule associated with the extracellular matrix when associated with each other.

[0429] Item 34. The kit or composition of any one of items 1 to 16, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding to a T cell, a macrophage or a natural killer cell when associated with each other.

[0430] Item 35. The kit or composition of Item 34, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to T cells when associated with each other.

[0431] Item 36. The kit or composition of Item 35, wherein the first complementary binding domain and the second complementary binding domain, when bound to each other, are capable of binding to CD3, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS).

[0432] Item 37. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding CD3 when combined together.

[0433] Item 38. The kit or composition of Item 37, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of muromonabin, otelizumab, telizumab, vesilizumab, foralizumab, SP34, or blinatumomab.

[0434] Item 39. The kit or composition of Item 36, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding to PD-1 when bound together.

[0435] Item 40. The kit or composition of Item 39, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of pembrolizumab or nivolumab.

[0436] Item 41. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding CTLA-4 when combined together.

[0437] Item 42. The kit or composition of Item 41, wherein the first and second supplementary binding domains comprise all or a portion of VH and / or VL of ipilimumab.

[0438] Item 43. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding TIM-3 when bound together.

[0439] Item 44. The kit or composition of Item 43, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of TSR-022 or Sym023.

[0440] Item 45. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding LAG-3 when combined together.

[0441] Item 46. The kit or composition of Item 45, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of BMS-986016.

[0442] Item 47. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to a KIR when combined together.

[0443] Item 48. The kit or composition of Item 47, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of lirelizumab.

[0444] Item 49. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding CD28 when bound together.

[0445] Item 50. The kit or composition of Item 49, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of cilalizumab.

[0446] Item 51. The kit or composition of Item 36, wherein the first and second complementary binding domains are capable of binding CD137 when combined together.

[0447] Item 52. The kit or composition of Item 51, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of Urelimumab or Urelimumab.

[0448] Item 53. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding OX40 when bound together.

[0449] Item 54. The kit or composition of Item 53, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of PF-04518600 or BMS986178.

[0450] Item 55. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding CD27 when combined together.

[0451] Item 56. The kit or composition of Item 55, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of varililumab.

[0452] Item 57. The kit or composition of Item 36, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding GITR (TNFRSF18) when combined together.

[0453] Item 58. The kit or composition of Item 57, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of GWN323 or BMS-986156.

[0454] Item 59. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding TIGIT when paired together.

[0455] Item 60. The kit or composition of Item 59, wherein the first supplementary binding domain and the second supplementary binding domain comprise all or a portion of the VH and / or VL of OMP-313M32, MTIG7192A, BMS-986207, or MK-7684.

[0456] Item 61. The kit or composition of Item 36, wherein the first complementary binding domain and the second complementary binding domain are capable of binding ICOS when combined together.

[0457] Item 62. The kit or composition of Item 61, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of JTX-2011.

[0458] Item 63. The kit or composition of Item 34, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to macrophages when associated with each other.

[0459] Item 64. The kit or composition of Item 63, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to CSF1R when associated with each other.

[0460] Item 65. The kit or composition of Item 65, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of ematuruzumab or IMC-CS4.

[0461] Item 66. The kit or composition of Item 63, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding CD40 when associated with each other.

[0462] Item 67. The kit or composition of Item 66, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of CP-870,893.

[0463] Item 68. The kit or composition of Item 34, wherein the first complementary binding domain and the second complementary binding domain are capable of binding natural killer cells when associated with each other.

[0464] Item 69. The kit or composition of Item 68, wherein the first supplementary binding domain and the second supplementary binding domain are capable of binding CD16A when associated with each other.

[0465] Item 70. The kit or composition of Item 69, wherein the first and second supplementary binding domains comprise all or a portion of the VH and / or VL of NTM-1633 or AFM13.

[0466] Item 71. The kit or composition of any one of items 1 to 70, wherein the first supplementary binding domain and the second supplementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to the same antigen.

[0467] Item 72. The kit or composition of any one of items 1 to 70, wherein the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to different antigens on the same cell.

[0468] Item 73. The kit or composition of any one of items 1 to 70, wherein the first complementary binding domain and the second complementary binding domain, when bound to each other, and the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to different cells.

[0469] Item 74. The kit or composition of any one of Items 1 to 73, wherein the first immune cell binding domain and the second immune cell binding domain are capable of forming an Fv when not bound to the first and second complementary binding domains.

[0470] Item 75. The kit or composition of any one of Items 1 to 74, wherein the first and second complementary binding domains are capable of forming an Fv when not bound to the first and second immune cell binding domains.

[0471] Item 76. A kit or composition for treating cancer in a patient, the kit or composition comprising a first component, the 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; a first immune cell binding domain capable of having immune cell binding activity when bound to a second immune cell binding domain, wherein the second immune cell binding domain is not part of the first component, and wherein the first immune cell binding domain is a VH domain or a VL domain; a first inert binding partner for the first immune cell binding domain, wherein the first inert binding partner 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, wherein if the first immune cell binding domain is a VH domain, then the inert binding partner is a VL domain, and if the first immune cell binding domain is a VH domain, then the inert binding partner is a VL domain. wherein the first immune cell binding domain is a VL domain, then the inert binding partner is a VH domain; a protease cleavage site separating the first immune cell binding domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease expressed by the cancer or tumor microenvironment cells; or is co-localized to the cancer by a targeting portion that is an antibody or antigen-binding fragment thereof, the targeting portion binds (a) a tumor antigen expressed by the cancer and is the same or different as the first targeting portion and / or the second targeting portion in the agent, or binds (b) an antigen expressed by cells in the tumor microenvironment; and a first supplementary functional domain capable of immune cell binding, and a second component comprising a targeted immune cell binding agent, the targeted immune cell binding agent comprising a second targeting portion; a second immune cell binding domain; and optionally a second supplementary functional domain capable of immune cell binding.

[0472] Item 77. The kit or composition of Item 76, wherein the second component comprises a supplementary functional domain.

[0473] Item 78. The kit or composition of any one of items 76-77, wherein the complementary functional domain of the first component and / or the second component comprises a ligand of a receptor.

[0474] Item 79. The kit or composition of Item 78, wherein the supplementary functional domain is a latently active form of a member of the TGF-β family.

[0475] Item 80. The kit or composition of Item 78, wherein the supplementary functional domain of the first component and / or the second component comprises a cytokine.

[0476] Item 81. The kit or composition of Item 78, wherein the cytokine is a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

[0477] Item 82. The kit or composition of Item 80, wherein the supplementary functional domain of the first component and / or the second component comprises an attenuated cytokine.

[0478] Item 83. The kit or composition of Item 82, wherein the attenuated cytokine is a variant of IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or a member of the TNF superfamily.

[0479] Item 84. The kit or composition of any one of Items 76 to 83, wherein the second component further comprises a second inert binding partner for 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, wherein if the second immune cell binding domain is a VH domain, the inert binding partner is a VL domain, and if the second immune cell binding domain is a VL domain, the inert binding partner is The binding partner is a VH domain; and further, wherein a protease cleavage site separates the second immune cell binding domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell binding domain in the presence of a protease, wherein the protease is expressed by the cancer; or is co-localized to the cancer by a targeting moiety that is an antibody or antigen-binding fragment thereof, which binds (a) a tumor antigen expressed by the cancer and is the same or different from the first targeting moiety and / or the second targeting moiety in the agent, or binds (b) an antigen expressed by cells in the tumor microenvironment.

[0480] Item 85. The kit or composition of any one of Items 1-84, wherein the first component is not covalently bound to the second component.

[0481] Item 86. A molecule comprising the kit or composition of any one of Items 1 to 85, wherein the first component is covalently bound to the second component.

[0482] Item 87. The single molecule of Item 86, wherein the first component is covalently bound to the second component via a cleavable linker.

[0483] Item 88. The kit or composition of any one of Items 1 to 87, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to a T cell, a macrophage, or a natural killer cell when associated with each other.

[0484] Item 89. The kit or composition of Item 88, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to a T cell when associated with each other.

[0485] Item 90. The kit or composition of Item 89, wherein the first immune cell binding domain and the second immune cell binding domain, when bound to each other, are capable of binding to CD3, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS).

[0486] Item 91. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CD3 when associated with each other.

[0487] Item 92. The kit or composition of Item 91, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of muromonabin, otelizumab, telizumab, vesilizumab, foralizumab, SP34, or blinatumomab.

[0488] Item 93. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to the T cell receptor when associated with each other.

[0489] Item 94. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to PD-1 when bound together.

[0490] Item 95. The kit or composition of Item 94, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of pembrolizumab or nivolumab.

[0491] Item 96. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CTLA-4 when bound together.

[0492] Item 97. The kit or composition of Item 96, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of VH and / or VL of ipilimumab.

[0493] Item 98. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to TIM-3 when bound together.

[0494] Item 99. The kit or composition of Item 98, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of TSR-022 or Sym023.

[0495] Item 100. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding LAG-3 when combined together.

[0496] Item 101. The kit or composition of Item 100, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of VH and / or VL of BMS-986016.

[0497] Item 102. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to KIR when combined together.

[0498] Item 103. The kit or composition of Item 102, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of lirelizumab.

[0499] Item 104. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CD28 when combined together.

[0500] Item 105. The kit or composition of Item 104, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of VH and / or VL of cilalizumab.

[0501] Item 106. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CD137 when bound together.

[0502] Item 107. The kit or composition of Item 106, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of Urelimumab or Urelimumab.

[0503] Item 108. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding OX40 when combined together.

[0504] Item 109. The kit or composition of Item 108, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of PF-04518600 or BMS 986178.

[0505] Item 110. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CD27 when combined together.

[0506] Item 111. The kit or composition of Item 110, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of VH and / or VL of varizumab.

[0507] Item 112. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to GITR (TNFRSF18) when combined together.

[0508] Item 113. The kit or composition of Item 112, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of GWN323 or BMS-986156.

[0509] Item 114. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding TIGIT when combined together.

[0510] Item 115. The kit or composition of Item 114, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of OMP-313M32, MTIG7192A, BMS-986207, or MK-7684.

[0511] Item 116. The kit or composition of Item 90, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding ICOS when combined together.

[0512] Item 117. The kit or composition of Item 116, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of VH and / or VL of JTX-2011.

[0513] Item 118. The kit or composition of Item 88, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to macrophages when associated with each other.

[0514] Item 119. The kit or composition of Item 118, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to CSF1R when associated with each other.

[0515] Item 120. The kit or composition of Item 119, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of ematuruzumab or IMC-CS4.

[0516] Item 121. The kit or composition of Item 120, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding CD40 when associated with each other.

[0517] Item 122. The kit or composition of Item 121, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of CP-870,893.

[0518] Item 123. The kit or composition of Item 88, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to natural killer cells when associated with each other.

[0519] Item 124. The kit or composition of Item 123, wherein the first immune cell binding domain and the second immune binding domain are capable of binding CD16A when associated with each other.

[0520] Item 125. The kit or composition of Item 124, wherein the first immune cell binding domain and the second immune cell binding domain comprise all or a portion of the VH and / or VL of NTM-1633 or AFM13.

[0521] Item 126. The kit or composition of any one of Items 1-125, wherein the first targeting moiety and the second targeting moiety are different.

[0522] Item 127. The kit or composition of any one of Items 1-125, wherein the first targeting moiety and the second targeting moiety are the same.

[0523] Item 128. The kit or composition of any one of Items 1 to 127, wherein the first targeting moiety and / or the second targeting moiety comprises an antibody or an antigen-binding fragment thereof.

[0524] Item 129. The kit or composition of any one of Items 1 to 128, wherein the first targeting moiety and / or the second targeting moiety comprises a DNA aptamer, an RNA aptamer, an albumin, an apolipoprotein, a fibronectin, an ankyrin, a fynomer, an Obody, a DARPin, a knotin, an affimer, an atrimer, an anti-callin, an affilin, an affibody, a bicyclic peptide, a cys knot, FN3 (adnectin, centryrin, pronectin or TN3) or a Kunitz domain.

[0525] Item 130. The kit or composition of any one of Items 1-129, wherein the second targeting moiety binds to a tumor antigen expressed by the cancer.

[0526] Item 131. The kit or composition of Item 128, wherein the first targeting moiety and / or the second targeting moiety comprises an antibody or antigen-binding fragment thereof that binds to: α4-integrin; A33; ACVRL 1 / 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; CLDN18.2; 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; epithelial sialoprotein; FAP; FGFR1; FGFR2; FGFR3; FGFR4; fibronectin additional structure 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; TWEAKR; TYRP1; VANGL2; VEGF; VEGF-C; VEGFR-2; or VEGF-R2.

[0527] Item 132. The kit or composition of Item 128, wherein the first targeting moiety and / or the second targeting moiety comprises an anti-α4-integrin antibody; an anti-CD137 antibody; an anti-CCR4 antibody; an anti-CD123 antibody; an anti-CD133 antibody; an anti-CD138 antibody; an anti-CD19 antibody; an anti-CD20 antibody; an anti-CD22 antibody; an anti-CD33 antibody; an anti-CD38 antibody; an anti-CD40 antibody; an anti-CD49d antibody; an anti-CD52 antibody; an anti-CD70 antibody; an anti-CD74 antibody; an anti-CD79b antibody; an anti-CD80 antibody; an anti-CEA antibody; an anti-cMet antibody; an anti-Cripto antibody; an anti-CTLA-4 antibody; an anti-DLL3 antibody; an anti-TRAIL-2 / DR5 antibody; an anti-E-cadherin antibody; an anti-endoglin antibodies; anti-EpCAM antibodies; anti-epidermal growth factor receptor antibodies; anti-FGFR3 antibodies; anti-fibronectin extra domain B antibodies; anti-folate receptor 1 antibodies; anti-glypican 3 antibodies; anti-gp95 / 97 antibodies; anti-Her2 antibodies; anti-IGF-1R antibodies; anti-IL-13R antibodies; anti-IL-4 antibodies; anti-IL-6 antibodies; anti-MMP-9 antibodies; anti-MUC1 antibodies; anti-mucin core protein antibodies; anti-NGcGM3 antibodies; anti-P-cadherin antibodies; anti-PD-L1 antibodies; anti-p-glycoprotein antibodies; anti-PSCA antibodies; anti-PSMA antibodies; anti-SLAMF7 antibodies; anti-TRAIL-R1 / DR4 antibodies; anti-transferrin antibodies; anti-TROP-2 antibodies; or anti-VEGF antibodies or antigen-binding fragments thereof.

[0528] Item 133. The kit or composition of Item 128, wherein the first targeting moiety and / or the second targeting moiety comprises alemtuzumab, andecaliximab, atezolizumab, avelumab, BCD-100, bevacizumab, BGB-A317, blinatumomab, brentuximab, BU59, camrelizumab, caplotuximab, catumaxomab, cemiplimab, cetuximab, daratumumab, depatuximab, dinutuximab, DS-8201, durvalumab, edrecolomab, elotuzumab, G544, gemtuzumab, gebatumumab, GP1.4, hp67.6, IBI308, isbetumomab, Inotuzumab, ipilimumab, isatuximab, L19IL2, L19TNF, magetuximab, milvituximab, mogamurizumab, mositumomab, natalizumab, nesimumab, nivolumab, obinutuzumab, ofatumumab, oratuzumab, ocituzumab, panitumumab, PDR001, pembrolizumab, pertuzumab, polouzumab, rantuzumab, ramucirumab, rituximab, rovaccinumab, certolizumab pegol, SM3, TAK-164, tositumomab, trastuzumab, tremelimumab, utoximab, usulelumab, utomiliumab, XMAB-5574, or zolbetumumab.

[0529] Item 134. The kit or composition of any one of items 1 to 127, wherein the first targeting moiety and / or the second targeting moiety comprises 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.

[0530] Item 135. The kit or composition of Item 134, wherein the first targeting moiety and / or the second targeting moiety comprises the full-length sequence of 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.

[0531] Item 136. The kit or composition of Item 134, wherein the first targeting moiety and / or the second targeting moiety comprises a truncated form, analog, variant or derivative of 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.

[0532] Item 137. The kit or composition of any one of items 1 to 134, wherein the first targeting moiety and / or the 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.

[0533] Item 138. The kit or composition of any one of Items 1-129, wherein the second targeting moiety binds to an antigen expressed by a cell of the tumor microenvironment.

[0534] Item 139. The kit or composition of Item 138, wherein the tumor microenvironment cells are fibroblasts or macrophages.

[0535] Item 140. The kit or composition of Item 139, wherein the antigen expressed by fibroblasts is fibroblast activation protein.

[0536] Item 141. The kit or composition of Item 139, wherein the antigen expressed by macrophages is MAC-1 / CD11b or sideroflexin 3.

[0537] Item 142. A method of treating a cancer expressing a tumor antigen that binds a first targeting moiety and / or a second targeting moiety in a patient, the method comprising administering the composition of any one of items 1-141 to the patient.

[0538] Item 143. The method of Item 142, wherein the presence of infiltrating immune cells in the cancer is assessed prior to administering the composition.

[0539] Item 144. The method of any one of items 142-143, wherein the presence of a tumor antigen in the cancer is assessed prior to administering the composition.

[0540] Item 145. The method of any one of items 142-144, wherein the first targeting moiety and the second targeting moiety bind to the same antigen.

[0541] Item 146. The method of any one of items 142-144, wherein the first targeting moiety and the second targeting moiety bind different antigens.

[0542] Item 147. The method of any one of items 142-146, wherein the cancer expressing a tumor antigen that binds to the first targeting moiety and / or the second targeting moiety is any one of the following: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, kidney cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease, or a premalignant disease.

[0543] Item 148. A method of targeting immune cells to a cancer in a patient that expresses a tumor antigen that binds both a first targeting moiety and / or a second targeting moiety, the method comprising administering to the patient the composition of any one of items 1-141.

[0544] Item 149. A method of targeting immune cells to a cancer expressing two tumor antigens in a patient, wherein one tumor antigen is bound to a first targeting moiety and one tumor antigen is bound to a second targeting moiety, the method comprising administering the composition of any one of items 1-141 to the patient.

[0545] Item 150. A method of delivering cytokines to immune cells in a patient, the method comprising administering to the patient the composition of any one of items 76 to 147, wherein the first supplementary functional domain and / or the second supplementary functional domain of TWICE comprises a member of the IL-2, IL-7, IL-12, IL-15, GM-CSF, IFN-α, IFN-γ, or TNF superfamily.

[0546] V. Support Form

[0547] The following supporting forms have been mentioned in the above application.

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[0586] Example

[0587] Example 1. TWICE blocks two inhibitory pathways in immune cells

[0588] TWICE can be developed to act on a range of cancers and immune cells. These examples are provided to show how one skilled in the art can generate TWICE with specific functions.

[0589] For example, TWICE can be generated with a synergistic combination of two checkpoint inhibitory antibodies.

[0590] As a first step, one skilled in the art will first select a targeting moiety that is suitable for a given cancer. The targeting moiety can be selected based on the literature on a given type of cancer and the prevalence of certain antigens. Alternatively, the antigens expressed by the cancer in a particular patient can be assessed. For example, rituximab (anti-CD20 antibody) can be used to target CD20-positive lymphomas, and cetuximab (anti-EGFR antibody) can be used to target certain solid tumors.

[0591] TWICE allows the use of two targeting moieties. One skilled in the art can select two targeting moieties so that both the first component and the second component are targeted to a given cancer. Using CA125 for targeting one component and EpCAM for targeting the other component can target ovarian cancer.

[0592] After selecting an appropriate targeting moiety, one skilled in the art can determine what types of immune cell binding domains and complementary binding domains, when paired, are likely to have the greatest therapeutic efficacy.

[0593] Figure 1 shows how TWICE can employ two cancer cell targeting domains to allow activation through two signaling pathways on immune cells.

[0594] To enhance the anti-tumor immune response, it may be desirable to produce TWICE that inhibits both checkpoint molecules, thereby enhancing the anti-cancer immune response compared to inhibiting either checkpoint molecule alone.

[0595] For example, a TWICE can be generated in which the first component comprises the VH of ipilimumab (anti-CTLA4 antibody) as the immune cell binding domain and the VL of nivolumab (anti-PD-1 antibody) as the complementary binding domain. The second component of this TWICE can then comprise the VL of ipilimumab (anti-CTLA4 antibody) as the immune cell binding domain and the VH of nivolumab (anti-PD-1 antibody) as the complementary binding domain. The function of the TWICE is not affected by which component has the VH or VL of a given antibody, as long as one component has the VH and the other has the VL.

[0596] By generating TWICE in this way, both the first and second components of TWICE can be targeted to cancer. At the cancer site, the VH and VL of ipilimumab and the VH and VL of nivolumab can be paired based on the close proximity of the first and second components. This will allow TWICE to block both immune checkpoints after domain pairing and lead to robust immune cell activation.

[0597] Example 2. TWICE stimulates two pathways in immune cells

[0598] TWICE can be developed that (when the immune cell binding domain and the complementary binding domain are paired) stimulates two pathways in the immune cell to mediate a robust anti-cancer effect. For example, the cancer can be breast cancer that expresses EGFR.

[0599] For any of these embodiments, the second targeting moiety can be selected to target cells of the tumor microenvironment. In this embodiment, the second targeting moiety can be selected to target tumor-associated macrophages (TAMs). Thus, while the first targeting moiety (in the first component) can bind to an antigen expressed by the tumor, the second targeting moiety (in the second component) can bind to an antigen expressed by TAMs. For targeting breast cancer expressing EGFR, the first targeting moiety can be cetuximab, and the second targeting moiety can be MAC-1 / CD11b.

[0600] Figure 2 Overview of TWICE where the first component is targeted to cancer cells and the second component is targeted to non-cancerous cells (in this example, TAMs). This type of TWICE will also only pair domains when cancer cells are in close proximity to TAMs. Pairing of immune cell binding domains and pairing of complementary binding domains can stimulate two pathways on immune effector cells.

[0601] For example, one skilled in the art may choose to activate both the CD3 e pathway and the CD137 pathway. To this end, he / she may generate TWICE, wherein the first component comprises the VH of SP34 (anti-CD3 antibody) and the VL of Utomidumab (anti-CD137 antibody), and the second component comprises the VL of SP34 (anti-CD3 antibody) and the VH of Utomidumab (anti-CD137 antibody).

[0602] By generating TWICE in this way, both the first and second components of TWICE can be targeted to cancer cells in close proximity to TAMs, such as those in the tumor stroma. The VH and VL of SP34 and the VH and VL of Utomitrumumab can then be paired based on the close proximity of the first and second components. This will allow TWICE to provide two positive stimuli to T cells after domain pairing.

[0603] In a manner similar to that described in this Example or Example 1, TWICEs can be generated to block inhibitory checkpoint molecules (e.g., through paired immune cell binding domains) and provide positive stimulation to the immune system (e.g., through paired complementary binding domains). These types of constructs can also robustly activate anti-cancer immune responses.

[0604] Example 3. TWICE activates two different immune cells at the cancer site

[0605] TWICE can combine the activities of two different immune effector cells. For example, TWICE can be generated to mediate T cell activation and macrophage activation.

[0606] Targeting moieties directed against antigens expressed by cancer cells can be selected as outlined in Example 1. In this particular example, both targeting moieties can bind to HER2 on breast cancer cells. The two targeting moieties can be the same, or bind to different epitopes on HER2. Thus, Figure 3 As shown in , the paired immune cell binding domains of TWICE can bind to a first effector cell (in this case a T cell), and the paired complementary binding domains can bind to a second effector cell (in this case a macrophage).

[0607] A person skilled in the art can choose to activate both the CD3 pathway in T cells and the CD40 pathway in macrophages. To do this, he / she can generate TWICE, wherein the first component comprises the VH of SP34 (anti-CD3 antibody) and the VL of CP-870,893 (anti-CD40 antibody), and the second component comprises the VL of SP34 (anti-CD3 antibody) and the VH of CP-870,893 (anti-CD40 antibody).

[0608] By creating TWICE in this way, the first and second components will be targeted to cancer cells. At the cancer cell site, the VH and VL of SP34 and the VH and VL of CP-870,893 can pair up based on the close proximity of the first and second components. This will allow TWICE to stimulate both T cells and macrophages at the cancer site after domain pairing.

[0609] In a similar manner, TWICE can be designed to activate a combination of T cells and NK cells at cancer sites.

[0610] Example 4. TWICE regulates immune cells and cancer cells

[0611] TWICE can also combine activities against immune cells and directly against cancer cells. For example, TWICE can be generated to mediate T cell activation and block immune checkpoint molecules expressed by cancer cells.

[0612] Two targeting moieties directed against a single antigen expressed by cancer cells, such as HER2 on breast cancer cells, can be selected as outlined in Example 3. Figure 4 As shown in , the paired immune cell binding domains of TWICE can bind effector cells (in this case, T cells), and the paired complementary binding domains can bind cancer.

[0613] A person skilled in the art can choose to activate CD3 in T cells and block PD-L1 in cancer cells. To do this, he / she can generate TWICE, wherein the first component comprises the VH of SP34 (anti-CD3 antibody) and the VL of avelumab (anti-PD-L1 antibody), and the second component comprises the VL of SP34 (anti-CD3 antibody) and the VH of avelumab (anti-PD-L1 antibody).

[0614] By creating TWICE in this way, both components will be targeted to cancer cells. There, the VH and VL of SP34 and the VH and VL of avelumab can pair based on the close proximity of the first and second components. This will allow TWICE to stimulate T cells in the tumor microenvironment after domain pairing and block immunosuppression mediated by PD-L1 expressed by cancer cells.

[0615] In a similar manner, TWICE can be designed to bind RANK or cell death-inducing antigens on cancer cells.

[0616] For example, one skilled in the art can choose to activate CD3 on T cells and directly induce cancer cell death. To do this, he / she can generate TWICE, in which the first component comprises the VH of SP34 (anti-CD3 antibody) and the VL of mapatumumab (anti-TRAIL-R1 / DR4 death receptor antibody), and the second component comprises the VL of SP34 (anti-CD3 antibody) and the VH of mapatumumab (anti-TRAIL-R1 / DR4 antibody). This will allow TWICE to stimulate T cells after domain pairing at the cancer cell site and directly trigger cancer cell death.

[0617] Therefore, TWICE can mediate direct anticancer effects and stimulate anticancer immune responses.

[0618] Example 5. TWICE containing a dimerization domain

[0619] In any of the constructs described in Examples 1-4, TWICE may further comprise a dimerization domain, such as Figure 6 As shown in . One dimerization domain of a pair can be connected to an immune cell binding domain, and the other dimerization domain of the pair can be connected to a complementary binding domain. For example, each component can comprise a pair of knob-into-hole dimerization sequences. One or both of the dimerization domains can be connected by a cleavable linker (referred to as a dimerization domain linker) such as a linker comprising an ADAM28 cleavage site (SEQ ID NO: 1).

[0620] In this way, the knob-into-hole dimerization domain of each component forces the pairing of the immune cell binding domain and the complementary binding domain of the individual components. After targeting to the tumor microenvironment by the targeting moiety, the cleavable linker connecting one or both dimerization domains can be cleaved, releasing the dimerization domain. In this way, the immune cell binding domain and / or the complementary binding domain will become available for pairing with their corresponding domain in the other component only in the tumor microenvironment.

[0621] Thus, the dimerization domain can be used to force the immune cell binding domain and the complementary binding domain of each component to bind outside the desired site of action in the tumor microenvironment and block domain pairing between components outside the tumor microenvironment. Conversely, in the tumor microenvironment, cleavage of the dimerization domain linker by a protease can allow domain exchange and activity to occur only at the tumor.

[0622] TWICEs can be designed where both components contain a dimerization domain, or where only one component contains a dimerization domain.

[0623] Example 6. TWICE containing complementary functional domains

[0624] Other TWICEs may contain complementary functional domains that are active when targeted to cancer cells, such as Figure 5 In this embodiment, the first component comprises attenuated IFN-α cytokine (see Pogue et al., PLoS ONE 11(9):e0162472 (2016)).

[0625] Both the first component and the second component may comprise an anti-EPCAM scFv. The first component may comprise an anti-CD3E VH domain, such as the VH domain of SP34 (i.e., an immune cell binding domain), connected to an inert binding partner VL domain from gantenerumab via a 25-mer linker with an MMP2 cleavage site (AIPVSLR; SEQ ID NO: 46). The second component may comprise an anti-CD3E VL domain, such as the VL domain of SP34 (i.e., an immune cell binding domain), connected to an inert binding partner VH domain from clone α-MUC1-1 antibody via a 25-mer linker with an MMP2 cleavage site (AIPVSLR; SEQ ID NO: 46).

[0626] In this way, the immune cell binding domains of the first and second components are masked unless cleaved by the tumor-associated protease MMP2. When the first and second components are targeted to EPCAM-positive tumors, the cleavage achieved by MMP2 can mediate the release of the inert binding domain (i.e., the inert binding partner). In this way, the VH and VL of SP34 can bind to and activate T cells at the cancer site. In addition, targeting attenuated IFN-α to cancer cells via EPCAM antibodies can lead to direct anti-cancer effects. This type of TWICE can mediate anti-cancer effects by attenuating cytokines and stimulating anti-cancer immune responses.

[0627] Similarly, TWICE can use IL-15 or attenuated IL-2 to regulate T cell activation.

[0628] In a similar manner, TWICE can be designed to include other attenuated cytokines as supplementary functional domains in the first component. TWICE can also be designed to include supplementary functional domains in both the first component and the second component to achieve an additive effect.

[0629] Example 7. TWICEs containing various dimerization domain linkers and various dimerization domains

[0630] To construct an example of a TWICE molecule, a well-established anti-EpCAM antibody targeting cancer cells was used. The EpCAM antibody solitonomab (Brischwein K et al. Mol Immunol. 43(8):1129-43 (2006)) was selected and used in the form of an antigen-binding fragment (Fab) comprising two chains, the heavy chain consisting of a heavy chain variable domain plus a CH1 domain, and the light chain consisting of a light chain variable domain and a kappa constant domain.

[0631] For initial testing of these TWICE molecules, the VH of the anti-CD3ε antibody SP34 was fused to the C-terminus of solitonumab Fab, and the VL of the anti-PDL1 antibody atezolizumab was fused to the C-terminus of the light chain of solitonumab Fab using flexible linkers that were 15 amino acids in length.

[0632] like Figure 6 As shown in , TWICE molecules can contain a dimerization domain that connects mismatched variable domains and prevents domain exchange unless TWICE is proteolytically activated in the tumor microenvironment. This dimerization domain can be constructed in a heterodimerization form to enhance the efficient formation of heterodimers between the VH domain and the VL domain and avoid the formation of homodimers. Several heterodimerization domains have been described in the literature. To test which heterodimerization domains are suitable for engineering TWICE, several TWICEs were constructed and expressed using various heterodimerization domains.

[0633] First, use Figure 7A The coiled-coil heterodimerization domain shown in [ 1 ] constructs TWICE. Coiled-coils have also been referred to as leucine zippers. Sequences of coiled-coil domains that have been engineered to form acidic and basic coils to form heterodimers are listed in US 20160002356A1 and US 8877893 B2. In this example, the heavy chain of the TWICE molecule is fused to the basic coil (SEQ ID NO: 201), and the light chain is fused to the acidic coil (SEQ ID NO: 202). One of ordinary skill in the art will appreciate that the heavy chain can also be fused to the acidic coil and the light chain to the basic coil.

[0634] Table 12 lists the dimerization domain joints included in the coiled-coil TWICE construct. These dimerization domain joints may contain sequences that can be cleaved by proteases. Protease cleavage will result in separation of the dimerization domain from the rest of the TWICE molecule, which allows for complementation of immune cell binding VH or VL domains between two TWICEs by domain exchange. In the examples listed in Table 12, several examples of dimerization domain joints are listed. In some instances, the joint contains a sequence that can be cleaved by matrix metalloproteinases in a tumor microenvironment. In other instances, the dimerization domain joint contains a protease site that can be cleaved by factor Xa (FXa), which can be used for artificial activation of TWICE molecules in vitro. In other words, the factor Xa cleavage site is used as a substitute for the protease cleavage site in the examples because factor Xa is convenient for use in a laboratory environment. In some examples listed in Table 12, the heavy chain and light chain of a TWICE molecule contain the same protease cleavage sequence in their dimerization domain linker, and in other examples, the heavy chain and light chain of a TWICE molecule contain different protease cleavage sequences in their dimerization domain linker.

[0635] In some examples listed in Table 12, TWICE contains a heterodimeric Fc as a dimerization domain. Here, the lysine repositioning strategy disclosed in WO2017106462A1 is used to construct TWICE with a hetero Fc dimerization domain. Table 12 shows the dimerization domain linkers used in the TWICE molecules TWICE191 and TWICE192. TWICE191 comprises a heavy chain of SEQ ID NO: 171 and a light chain of SEQ ID NO: 172, and TWICE192 comprises a heavy chain of SEQ ID NO: 173 and a light chain of SEQ ID NO: 174. These TWICEs contain the mutations S364K / K409L in the Fc of the heavy chain and K370S / F405K in the Fc of the light chain to promote the formation of a hetero Fc between the heavy chain and the light chain. Engineering TWICE with a heteroFc dimerization domain enables binding to the neonatal Fc receptor FcRn, which mediates recycling of the molecules and prolongs their serum half-life. These TWICE examples also contain the N297Q mutation in the Fc, which produces an aglycosylated molecule with impaired effector function.

[0636] In another example, the Fab constant domains (CH1 and CL) were used as dimerization domains to construct the TWICE molecule. To allow efficient assembly of the heavy and light chains of the TWICE molecule that also contained Fab as a cancer targeting moiety, the dimerization domains CH1 and CL were constructed as exchange Fab ( Figure 7A). Exchange Fab is a strategy to enable efficient assembly of the two light chains of a bispecific antibody construct by exchanging the constant domains in one of the Fabs so that VH is connected to CL and VL is connected to CH1 (Schaefer W et al., Proc Natl Acad Sci US A. 108(27): 11187-92(2011)). In the TWICE constructs (TWICE200 and TWICE204, see Table 13) containing exchange Fab dimerization domains, the VH of the immune cell linker is connected to the CL domain with a dimerization domain linker sequence, and the VL of the complementary linker is connected to the CH1 domain with a dimerization domain linker sequence. The factor Xa protease cleavage sequence (Ile-Glu-Gly-Arg) is included in all dimerization domain linkers. The complete sequence of TWICE200 comprises the heavy chain of SEQ ID NO: 177 and the light chain of SEQ ID NO: 178, and construct TWICE204 comprises the heavy chain of SEQ ID NO: 179 and the light chain of SEQ ID NO: 180.

[0637] In another example, TWICE was constructed using the IgE CH2 domain as a dimerization domain. IgE CH2 typically serves as a hinge domain in IgE, but this domain has been used to construct Fab-like molecules with similar binding properties by domain replacement (Cooke et al., mAbs.10(8):1248-1259(2018)). In TWICE189 and TWICE190, the IgE CH2 domain was fused to the immune cell linker VH domain and VL domain using the dimerization domain linker sequence given in Table 12. The dimerization domain linker contains a factor Xa cleavage sequence (Ile-Glu-Gly-Arg).

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[0639]

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[0641]

[0642] The TWICE proteins listed in Table 13 were expressed in short HEK293 cultures (30-50 ml) in shake flasks by co-transfection of the corresponding heavy and light chains. The expressed proteins were purified from the supernatant by FPLC using an IMAC column (Histrap Excel, GE) or, in the case of TWICE proteins containing Fc, a protein A column (MabSelect PrismA, GE). The purified proteins were analyzed by SDS-PAGE and showed bands at the expected molecular weight for all linkers and dimerization domains tested ( Figure 7B The expression levels of TWICEs with coiled-coil or heteroFc dimerization domains were the highest.

[0643] TWICE was further tested by cleavage with recombinant FXa protease. In a 15 μl reaction, approximately 1 μg of purified TWICE and 0.1 μg of FXa (New England Biolabs #_P8010) were incubated in a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl and 2 mM CaCl2 for 1 hour at ambient temperature. The reaction was terminated by adding 5 μl of 4X LDS sample buffer (Thermo Fisher NP0007) and heating to 95°C for 2 minutes. The samples were then separated by SDS PAGE to analyze the cleavage products ( Figure 7C ).

[0644] Samples TWICE193, TWICE191 and TWICE192 (Table 13) containing FXa cleavage sequences in both dimerization domain linkers, i.e. in the heavy chain as well as in the light chain, were completely cleaved by FXa ( Figure 7C TWICE117, which has a FXa cleavage site in the heavy chain dimerization domain linker and an MMP2 cleavage site in the light chain dimerization domain linker, was only partially cleaved ( Figure 7B). Constructs TWICE200, TWICE204, TWICE189, and TWICE190, which have FXa cleavage sites included in the linkers of the two dimerization domains, were not cleaved by the FXa protease under these conditions. This result suggests that the configuration of the dimerization domains (CH1 / CL or IgE CH2) in constructs TWICE200, TWICE204, TWICE189, and TWICE190 or the linker lengths of 5 to 8 residues in length (Table 12) were not conducive to protease cleavage in these particular constructs. However, the coiled-coil and hetero-Fc dimerization domains included in designs TWICE117, TWICE193, TWICE192, and TWICE193 were suitable for constructing TWICE. TWICE molecules containing these dimerization domains expressed well and could be activated by cleavage with recombinant proteases.

[0645] Example 8. TWICE with various binding moieties: Fv versus Fab

[0646] TWICE molecules were engineered with CD3 antibody SP34 and CTLA4 antibody ipilimumab, as well as containing hetero-Fc dimerization domains. In some instances, the anti-EpCAM antibody solitomab was used in Fab form (TWICE277 and TWICE278), and in other instances, this antibody was used in the form of a disulfide-stabilized Fv (dsFv, TWICE332 and TWICE335) by engineering a disulfide bond between residue 44 of VH and residue 100 of VL (relating to Kabat numbering). TWICE molecules were expressed and purified by protein A affinity chromatography as described above, and their ability to bind to EpCAM was tested in an ELISA binding assay. Recombinant soluble EpCAM-His6 protein (produced in-house) at 5 μg / ml was coated overnight on ELISA plates at 4°C, and the plates were blocked with PBS containing 1% BSA. Serial dilutions of TWICE were bound to EpCAM and bound TWICE was detected using a mouse anti-human Fc secondary antibody (JDC-10 Abcam, ab99759). Figures 8A to 8B ) showed that TWICE molecules with Fab or dsFv targeting moieties bound to antigen with similar binding affinities, with the KD for dsFv TWICE ranging from 1.04 nM to 1.65 nM and the KD for Fab TWICE ranging from 1.21 nM to 1.22 nM. Therefore, TWICE molecules can be constructed with different targeting moieties, such as Fab or dsFv, to target cells in the tumor microenvironment.

[0647] Example 9. Testing CD3 / CTLA-4 TWICE in a Bridging ELISA

[0648] To test the domain exchange and generation of functional paratopes (ie, paired VH / VL domains) achieved by TWICE pairs, a bridging ELISA assay was developed that measures the ability of TWICE pairs to simultaneously bind target antigens and immune cell engager molecules. Figure 9A The schematic diagram in Figure 1 illustrates the principle of this bridging ELISA. Briefly, an ELISA plate is coated overnight with the target antigen (recombinant soluble EpCAM, black hexagons) at a concentration of 5 μg / ml. TWICE molecules can be activated by cleavage with FXa as described above and then added to the plate in serial dilutions. TWICE can bind to the target antigen via their target binding moieties, which are located in the Figure 9A Fab (4 grey ovals) are drawn in the figure. Binding to the target antigen (EpCAM) on the plate brings the TWICE into close proximity, which allows the immune cell binding domain and the complementary binding domain to undergo domain exchange and form a complete immune cell binding paratope ( Figure 9A By binding to antigens such as CD3 ( Figure 9A Functional complementation was measured by incubating with the striped triangles in Figure 1. Antigen was provided as an Fc fusion protein (murine IgG2a Fc) and was therefore detected with an anti-mouse Fc secondary antibody. For clarity, Figure 9A This detection step using secondary antibodies is omitted from the schematic diagram.

[0649] Figure 9B and Figure 9C Examples of bridging ELISAs measuring functional complementation achieved by domain exchange of TWICE pairs are shown. In these examples, TWICE277 and 278 (as described in Table 13) were used, which contain the immune cell binding antibody SP34 that binds to CD3ε and the complementary antibody ipilimumab that binds to CTLA-4. The VH of the CD3 antibody and the VL of the CTLA-4 antibody are contained in TWICE277, and the VL of the anti-CD3 antibody and the VH of the anti-CTLA-4 antibody are contained in TWICE278. These examples of TWICE molecules also contain a hetero Fc dimerization domain and a FXa cleavage site in the linker of the two dimerization domains. Figure 9B Results from a CD3 bridging ELISA measuring the ability of TWICE to bind CD3 are shown, and Figure 9CThe results of the CTLA-4 bridging ELISA measuring the ability of TWICE to bind to CTLA-4 are shown. In this experiment, even when activated by cleavage with FXa, single TWICE (277 or 278 alone) did not show binding to CD3 or to CTLA-4. In addition, when TWICE 277 and 278 were combined but not activated by cleavage with FXa, TWICE did not show binding to CD3 or CTLA-4. Only when TWICE 277 and 278 were combined and cleaved by FXa to remove the dimerization domain did they show binding to CD3 or CTLA-4 in a dose-dependent response ( Figure 9B and Figure 9C These results demonstrate that when both TWICEs are bound to a target antigen, the TWICE molecule can undergo domain swapping and generate functional immune cell-binding domains. Furthermore, the dimerization domains of these TWICEs act as a locking mechanism to prevent domain swapping of TWICEs, as intended, unless the dimerization domains are removed by protease cleavage.

[0650] FIG. 10A to FIG. 10B Another example of a CD3 / CTLA-4 TWICE molecule tested in a bridging ELISA experiment is shown. TWICE281 and 282 used in this experiment contain the same EpCAM targeting antibody and CD3 and CTLA-4 antibodies as TWICE277 and TWICE278 described above, but differ in that they contain a coiled-coil dimerization domain. In addition, TWICE281 and 282 contain a FXa cleavage site in the heavy chain and an MMP2 cleavage sequence in the light chain, and the molecules can therefore be activated by cleavage by either FXa or MMP2. Similar to the previous experiments, TWICE277 and 278 showed a dose-response binding curve only when activated by cleavage with FXa ( Figure 10A and Figure 10B Without FXa activation, TWICE does not bind CD3 or CTLA-4. Therefore, both hetero-Fc and coiled-coil dimerization domains are suitable for engineering TWICE. When an extended serum half-life of TWICE is desired, hetero-Fc may be preferred.

[0651] Example 10. CD3 / CD28 TWICE

[0652] Figures 11A to 11BAn example of TWICE constructed from the CD3 antibody SP34 and the co-stimulatory anti-CD28 antibody cilalizumab tested in a bridging ELISA experiment is shown (TWICE 394 and 395 from Table 13). These examples of TWICE were again engineered using hetero-Fc dimerization domains and FXa cleavage sites in both the heavy and light chain linkers. Similar to the previous examples, TWICE394 and TWICE395 showed binding to the immune cell antigens CD3 and CD28 only when they were combined and activated by cleavage ( Figure 11A and Figure 11B Single TWICE did not show binding to any immune cell antigen. Cleavage is again necessary for domain exchange and binding, as evidenced by the fact that uncleaved TWICE did not show any bridging activity ( Figure 11A and Figure 11B gray squares in the .

[0653] Example 11. CD28 / 4-1BB TWICE

[0654] FIG. 12A to FIG. 12B An example of TWICE constructed with the antibodies Cilalizumab and Urelizumab, which bind to the co-stimulatory molecules CD28 and CD137 (4-1BB), is shown. Using the variable domains of Cilalizumab and Urelizumab and containing hetero Fc dimerization domains and FXa cleavage sites in two linkers, TWICE400 and 401 (as shown in Table 13) were constructed. When these molecules were tested in a bridging ELISA format, when the construct was pre-cleaved with FXa and both TWICEs were present, TWICE showed binding to CD28 ( Figure 12A ) and 4-1BB( Figure 12B Neither TWICE binds to CD28 or 4-1BB alone.

[0655] Example 12. CTLA-4 / PD-L1 TWICE

[0656] Figure 13 An example of TWICE constructed with ipilimumab and atezolizumab, antibodies that bind to the checkpoint molecules CTLA-4 and PD-L1, respectively, is shown. TWICE283 contains the VH of ipilimumab and the VL of atezolizumab, and the complementary TWICE284 contains the VH of atezolizumab and the VL of ipilimumab, as shown in Table 13. These TWICEs were designed with a coiled-coil dimerization domain and a FXa cleavage site in the heavy chain. When these TWICEs were tested in an EpCAM / PD-L1 bridging ELISA, TWICE 284 exhibited significant binding to PD-L1 ( Figure 13), although this TWICE contained only the VH of atezolizumab but lacked the cognate VL of atezolizumab. This result suggests that the VH of atezolizumab is sufficient to mediate antigen binding.

[0657] This insight is consistent with the fact that the crystal structure of atezolizumab in complex with PD-L1 (Lee HT et al. Sci Rep 7:5532-5532 (2017)) reveals that the VL of the antibody does not make significant contact with the antigen and may therefore be dispensable or replaceable for antigen binding. Therefore, atezolizumab is an example of an antibody that is not suitable for constructing TWICE, at least not in combination with ipilimumab, due to the fact that atezolizumab retains its binding properties in the VH domain when the paratope (i.e., VH / VL pair) is split in TWICE.

[0658] However, if 9A to 12B As shown in , ipilimumab, SP34, cilalizumab, and usrelizumab are exemplary antibodies that exhibit no binding when the paratope is resolved, and such antibodies are therefore suitable for constructing TWICE.

[0659] Example 13. Immune cell activation achieved by TWICE

[0660] T cell redirection achieved by the TWICE construct was tested in an in vitro T cell activation assay. Briefly, colorectal cancer cells (HCT-15) were seeded in 96-well plates at a density of 5,000 cells / well and allowed to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were added at an effector-target ratio of 10:1 and the cells were treated with serial dilutions of the TWICE molecule. In some experiments, the TWICE molecule was pre-activated by cleavage with FXa protease (NEB #P8010L) before addition to the cells.

[0661] To quantify T cell activation, secreted interferon gamma was detected in the culture medium 24 hours after addition of TWICE using an IFNγ ELISA kit (Invitrogen 88-7316-88). Target cell killing was determined 48 hours after the start of treatment using a cytotoxicity assay (CytoTox96, Promega) that quantitatively measures lactate dehydrogenase (LDH) from lysed cancer cells. Target cell killing was expressed as Figures 14A to 16 Specific lysis (%) in the dose-response curves shown in , corresponds to the fraction of LDH released compared to cells lysed with lysis buffer and with background LDH release from untreated cells subtracted.

[0662] FIG. 14A to FIG. 14BResults of cell killing assays performed with TWICE277 and 278, as well as TWICE281 and 282, which have been previously tested in bridging ELISAs, are shown. As described above, these TWICEs contain the anti-CD3ε adapter antibody SP34 and the CTLA-4 antibody ipilimumab. TWICE277 and 278 were constructed with hetero-Fc dimerization domains linked to either VH or VL via a linker GGGIEGRGGG (SEQ ID NO: 206), which contained the FXa cleavage sequence Ile-Glu-Gly-Arg. In contrast, TWICE281 and 282 contained coiled-coil dimerization domains with non-identical linkers in the heavy and light chains ( Figure 14A These TWICEs were constructed using a FXa cleavage site in the heavy chain linker GGGGSIEGRGGGGS (SEQ ID NO: 203) and a MMP2 cleavage site in the light chain linker GGPLGVRGKGGGS (SEQ ID NO: 204). LDH release data from this experiment ( Figure 14B ) demonstrated that both TWICE pairs induced robust killing of cancer cells by PBMCs when TWICE was pre-activated by FXa prior to addition to cells. TWICE277 and 278 did not induce this killing response without pre-cleavage, confirming that cleavage is required for TWICE activation and domain exchange, and consistent with data obtained in bridging ELISA experiments with these molecules ( Figures 9B to 9C ) are consistent. However, in the bridging ELISA, activation by FXa is also required ( FIG. 10A to FIG. 10B ) readily induced killing of HCT15 cells by PBMCs in a dose-dependent manner even when TWICE was not preactivated.

[0663] These results demonstrate that TWICE can be activated by cancer cells that cleave the MMP2 site in the light-linked linker. In summary, this experiment confirms that: (1) TWICE can efficiently engage immune cells to kill cancer cells; (2) proteolytic cleavage is required for TWICE activation; and (3) proteases produced by cancer cells can activate TWICE.

[0664] 15A to 15D Lysis assays performed with TWICE 285 and TWICE 286, which also contained the anti-CD3ε engager antibody SP34 and the CTLA-4 antibody ipilimumab, are shown ( Figure 15A ), T cell activation assay ( Figure 15B ) and killing assays ( FIG. 15C to FIG. 15DThese examples of TWICE were designed with FXa cleavage sites in the dimerization domain linker GGGGSIEGRGGSGGGS (SEQ ID NO: 205) in both the heavy and light chains, which was longer than the above dimerization domain linker. FIG. 14A to FIG. 14B These examples of TWICE also contain a coiled-coil dimerization domain.

[0665] When TWICE285 was cleaved with FXa, the molecule produced the expected fragments on SDS-PAGE ( Figure 15A ), thus showing that TWICE can be preactivated with FXa. Figure 15B and Figure 15C The cytokine release and killing data shown in confirm that the TWICE molecules TWICE285 plus TWICE286 are able to engage and redirect PBMCs against HCT-15 cells when TWICE is activated by cleavage with FXa protease. Neither TWICE285 nor TWICE286 alone induces interferon gamma release or cancer cell killing by T cells.

[0666] However, when these TWICEs were tested together in an LDH release assay without pre-lysis, the molecules induced cancer cell lysis independent of pre-activation by FXa ( Figure 15D This result differs from the above TWICE examples that required cleavage to achieve activation in the bridging ELISA and T cell activation assays. Therefore, the data confirm that the design of the dimerization domain and linker length is relevant to TWICE activation. The specific longer linker GGGGSIEGRGGSGGGS (SEQ ID NO: 205) of TWICE285 and TWICE286, combined with the coiled-coil dimerization domain present in these constructs, does not prevent domain swapping in the absence of cleavage.

[0667] Example 14. Co-stimulatory TWICE

[0668] Next, TWICE394 and TWICE395 ( Figure 16 ). As described above, these TWICEs are constructed by the anti-CD28 antibody cilalizumab and the anti-CD3ε antibody SP34. Figures 11A to 11B As shown in , these molecules can bind to CD3 and CD28, which provides co-stimulatory signals to trigger T cell activation. When these TWICEs were tested in a T cell killing assay, TWICE394 and 395 induced robust killing of HCT-15 cancer cells after pre-activation with FXa, but showed no activity without prior lysis.

[0669] To test the stimulating signal obtained by TWICE engagement CD28 and CD3, a scheme for exhausting T cells before testing TWICE activity was developed. For T cell activation and exhaustion, prostate cancer cells (PC-3) were seeded in 6-well plates at a density of 150,000 cells / well and allowed to adhere overnight. Peripheral blood mononuclear cells (PBMC) were added at an effect-target ratio of 10: 1, and cells were processed at a concentration of 100 nM using a two-component T cell adapter based on SEQ ID NO: 168 and 169 of US10035856B2. This two-component system works similarly to CD3 bispecific antibodies because T cells are engaged to kill cancer cells. However, with respect to pre-activation, it is superior to conventional bispecific antibodies because the molecule cannot bind the CD3 in the solution and therefore can be easily removed by washing PBMC. PBMC was analyzed 48 hours after pre-activation by flow cytometry, thereby revealing that the robust upregulation of activation and exhaustion markers on treatment-induced CD3+ cells was observed. Figure 17A ), thus demonstrating pre-activation and exhaustion. Compared to unstimulated PBMCs, such pre-activated immune cells may be more reflective of immune cells in the tumor microenvironment, as tumor-infiltrating lymphocytes are known to be exhausted and show increased expression of exhaustion markers (Baitsch L et al. Trends Immunol. 33(7):364-72 (2012)).

[0670] Preactivated PBMCs were then used in a redirection assay against colorectal cancer cells (HCT-15) as described above to test the potency of TWICE molecules to trigger T cell activation, except that the effector-target ratio was modified to 1:4 in this assay. Figure 17BThe results of this T cell activation assay are shown. TWICE394 and TWICE395, which bind to CD3 and CD28, induce robust activation of exhausted T cells, as measured by IFNγ secretion. TWICE277 and TWICE278, which bind to CD3 and CTLA-4, also induce IFNγ secretion, although at lower levels than TWICE394 and TWICE395. A pair of control TWICEs containing CD3 antibody VH and VL domains paired with non-functional VH and VL domains (SEQ ID NOs: 199 and 200) were also included in this assay. In this redirection experiment performed with exhausted T cells, the control TWICE431 plus TWICE432 produced low levels of interferon gamma. In summary, these results are consistent with recent publications showing that co-stimulatory activation of CD28 and CD3 achieved by trispecific antibodies produces T cell activation superior to either stimulation alone, and can achieve a stronger anti-tumor immune response (Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer (published online on November 18, 2019) doi: 10.1038 / s43018-019-0004-z). Consistent with this observation, the data of the present invention show that TWICE can strongly stimulate exhaustion of T cells, and the cumulative effect of co-stimulation or checkpoint blockade can induce a stronger response than CD3 engagement alone. However, in contrast to the disclosed trispecific antibodies, TWICE can deliver these co-stimulatory signals in an activatable manner.

[0671] Example 15. TWICE to engage NK cells via CD16A

[0672] In another example, TWICE is engineered to engage natural killer (NK) cells using antibodies against CD16A. The anti-CD16A antibody used to construct these TWICE molecules has been evaluated in a clinical trial (NCT01221571) in a molecule AFM13 that is a bispecific antibody targeting CD30 and CD16A. For evaluating this antibody in a TWICE pair, the anti-CD16A antibody was combined with the anti-CD3 antibody OKT3 to produce TWICE molecules TWICE440 and TWICE441 that include a hetero Fc dimerization domain and a FXa cleavage site in the dimerization domain linker.

[0673] The TWICE molecules were first tested in a CD16A bridging ELISA similar to the bridging ELISA experiment described above. TWICE440 and 441 showed the expected binding to CD16A only when both TWICEs were present and pre-activated with FXa protease ( Figure 18A ). As seen with other TWICE molecules, pre-cleavage of TWICE is necessary, and uncleaved TWICE does not exhibit binding to CD16A in this bridging ELISA assay. In addition, single TWICE molecules do not show binding to CD16A even when they are pre-cleaved by FXa. Therefore, the anti-CD16A antibody derived from AFM13 is suitable for use in TWICE because the split paratope halves (i.e., VH and VL) do not bind antigen when they are separated in TWICE.

[0674] TWICE440 and 441 were then tested in a redirection assay using HCT15 colon cancer cells and NK cells. As described above for the redirection assay performed with PBMCs, cancer cells were seeded in 96-well plates at 5000 cells / well and allowed to adhere overnight. Then, purified NK cells (Stem Cell Technologies, catalog number 70036) were added to the cancer cells at an effector-target ratio of 20: 1, and pre-activated TWICE was added to the wells at various concentrations. Using a cytotoxicity assay (CytoTox96, Promega) that quantitatively measures LDH from lysed cancer cells, target cell killing was determined by measuring LDH release 24 hours after adding TWICE. The results of this NK cell redirection assay are shown in Figure 18B In this experiment, a control bispecific antibody (CTRL406) targeting EpCAM and CD16a induced LDH release from cancer cells, demonstrating killing by NK cells.

[0675] A control bispecific antibody targeting EpCAM and CD3 also induced LDH release, but to a lesser extent than the CD16a / EpCAM bispecific antibody, likely due to the presence of some NKT cells. TWICE440 and 441 induced robust NK redirection and cancer cell killing, similar to the amount of killing observed with the CD16a / EpCAM bispecific antibody, demonstrating that TWICE molecules can be engineered to engage NK cells via CD16a antibodies. Engaging NK cells via anti-CD16 antibodies can induce cancer cell killing.

[0676] equivalent

[0677] The foregoing written description is considered sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode contemplated by the inventors. However, it should be understood that no matter how detailed the foregoing may appear in the text, the embodiments can be implemented in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0678] As used herein, the term approximately refers to a numerical value, including, for example, an integer, a fraction, and a percentage, whether or not explicitly indicated. The term approximately generally refers to a range of numerical values ​​(e.g., + / - 5-10% of the stated range) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). When a term such as at least and approximately is placed before a list of numerical values ​​or ranges, the term modifies all values ​​or ranges provided in the list. In some cases, the term approximately may include a numerical value that is rounded to the nearest significant figure. Sequence Listing <110> REVITOPE LIMITED <120> Dual immune cell adapter <130> 01131-0023-00PCT <150> US 62 / 780,770 <151> 2018-12-17 <160> 215 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 1 Lys Pro Ala Lys Phe Phe Arg Leu 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 2 Asp Pro Ala Lys Phe Phe Arg Leu 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 3 Lys Pro Met Lys Phe Phe Arg Leu 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 4 Leu Pro Ala Lys Phe Phe Arg Leu 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 5 Leu Pro Met Lys Phe Phe Arg Leu 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 6 Lys Pro Ala Met Phe Phe Arg Leu 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 7 Tyr Pro Ala Lys Phe Phe Arg Leu 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 8 Lys Trp Ala Lys Phe Phe Arg Leu 1 5 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 9 Asp Pro Met Lys Phe Phe Arg Leu 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 10 Asp Pro Ala Met Phe Phe Arg Leu 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 11 Asp Pro Met Met Phe Phe Arg Leu 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 12 Lys Met Ala Met Phe Phe Arg Leu 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 13 Lys Met Ala Met Phe Phe Ile Met 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 14 Lys Pro Ala Met Phe Phe Ile Met 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 15 Leu Pro Ala Met Phe Phe Arg Leu 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 16 Leu Pro Met Met Phe Phe Arg Leu 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 17 Leu Met Ala Met Phe Phe Arg Leu 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 18 Leu Met Ala Met Phe Phe Ile Met 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 19 Leu Pro Ala Met Phe Phe Ile Met 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 20 Leu Pro Ala Met Phe Phe Tyr Met 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> twenty one Lys Pro Met Met Phe Phe Arg Leu 1 5 <210> twenty two <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> twenty two Lys Pro Ala Lys Phe Phe Tyr Met 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> twenty three Lys Pro Ala Lys Phe Phe Ile Met 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> twenty four Ile Pro Met Lys Phe Phe Arg Leu 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 25 Ile Pro Ala Met Phe Phe Arg Leu 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 26 Ile Pro Met Met Phe Phe Arg Leu 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 27 Ile Met Ala Met Phe Phe Arg Leu 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 28 Ile Met Ala Met Phe Phe Ile Met 1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 29 Ile Pro Ala Met Phe Phe Ile Met 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> ADAM28 cleavage site <400> 30 Ile Pro Ala Met Phe Phe Tyr Met 1 5 <210> 31 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 31 Phe Arg 1 <210> 32 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 32 Phe Lys 1 <210> 33 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 33 Val Ala 1 <210> 34 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 34 Val Arg 1 <210> 35 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <220> <221> misc_feature <222> (2)..(2) <223> Xaa is citrulline <400> 35 Val Xaa 1 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 36 His Leu Val Glu Ala Leu Tyr Leu 1 5 <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 37 Ser Leu Leu Lys Ser Arg Met Val Pro Asn Phe Asn 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 38 Ser Leu Leu Ile Ala Arg Arg Met Pro Asn Phe Asn 1 5 10 <210> 39 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 39 Lys Lys Phe Ala 1 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 40 Ala Phe Lys Lys 1 <210> 41 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin B cleavage site <400> 41 Gln Gln Gln 1 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin D cleavage site <400> 42 Pro Arg Ser Phe Phe Arg Leu Gly Lys 1 5 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin D cleavage site <400> 43 Ser Gly Val Val Ile Ala Thr Val Ile Val Ile Thr 1 5 10 <210> 44 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Cathepsin K cleavage site <400> 44 Gly Gly Pro 1 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> MMP1 cleavage site <400> 45 Ser Leu Gly Pro Gln Gly Ile Trp Gly Gln Phe Asn 1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MMP2 cleavage site <400> 46 Ala Ile Pro Val Ser Leu Arg 1 5 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> MMP2 cleavage site <400> 47 Ser Leu Pro Leu Gly Leu Trp Ala Pro Asn Phe Asn 1 5 10 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP2 cleavage site <400> 48 His Pro Val Gly Leu Leu Ala Arg 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP2 cleavage site <400> 49 Gly Pro Leu Gly Val Arg Gly Lys 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP2 cleavage site <400> 50 Gly Pro Leu Gly Leu Trp Ala Gln 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP3 cleavage site <400> 51 Ser Thr Ala Val Ile Val Ser Ala 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP7 cleavage site <400> 52 Gly Pro Leu Gly Leu Ala Arg Lys 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP7 cleavage site <400> 53 Arg Pro Leu Ala Leu Trp Arg Ser 1 5 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> MMP7 cleavage site <400> 54 Ser Leu Arg Pro Leu Ala Leu Trp Arg Ser Phe Asn 1 5 10 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> MMP2 / 9 cleavage site <400> 55 Gly Ile Leu Gly Val Pro 1 5 <210> 56 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP2 / 9 cleavage site <400> 56 Gly Pro Leu Gly Ile Ala Gly Gln 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP9 cleavage site <400> 57 Ala Val Arg Trp Leu Leu Thr Ala 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MMP9 cleavage site <400> 58 Pro Leu Gly Leu Tyr Ala Leu 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> MMP9 cleavage site <400> 59 Gly Pro Gln Gly Ile Ala Gly Gln Arg 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP9 cleavage site <400> 60 Lys Pro Val Ser Leu Ser Tyr Arg 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP11 cleavage site <400> 61 Ala Ala Ala Thr Ser Ile Ala Met 1 5 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP11 cleavage site <400> 62 Ala Ala Gly Ala Met Phe Leu Glu 1 5 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> MMP13 cleavage site <400> 63 Gly Pro Gln Gly Leu Ala Gly Gln Arg Gly Ile Val 1 5 10 <210> 64 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> MMP14 cleavage site <400> 64 Pro Arg His Leu Arg 1 5 <210> 65 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> MMP14 cleavage site <400> 65 Pro Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu Gly 1 5 10 <210> 66 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MMP14 cleavage site <400> 66 Pro Arg Ser Ala Lys Glu Leu Arg 1 5 <210> 67 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> PSA / KLK3 <400> 67 His Ser Ser Lys Leu Gln 1 5 <210> 68 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> PSA / KLK3 <400> 68 Ser Ser Lys Leu Gln 1 5 <210> 69 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> KLK4 <400> 69 Arg Gln Gln Arg 1 <210> 70 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> TMPRSS2 <400> 70 Gly Gly Arg 1 <210> 71 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> legume protein <400> 71 Ala Ala Asn 1 <210> 72 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> ST14 (matrix protease) <400> 72 Gln Ala Arg 1 <210> 73 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> C1s cleavage site <400> 73 Tyr Leu Gly Arg Ser Tyr Lys Val 1 5 <210> 74 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> C1s cleavage site <220> <221> misc_feature <222> (6) <223> Xaa can be any naturally occurring amino acid <400> 74 Met Gln Leu Gly Arg Xaa 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> MASP2 cleavage site <400> 75 Ser Leu Gly Arg Lys Ile Gln Ile 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> C2a and Bb cleavage sites <400> 76 Gly Leu Ala Arg Ser Asn Leu Asp Glu 1 5 <210> 77 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> uPa cleavage site <400> 77 Thr Tyr Ser Arg Ser Arg Tyr Leu 1 5 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> uPa cleavage site <400> 78 Lys Lys Ser Pro Gly Arg Val Val Gly Gly Ser Val 1 5 10 <210> 79 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> uPa cleavage site <400> 79 Asn Ser Gly Arg Ala Val Thr Tyr 1 5 <210> 80 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> uPa cleavage site <400> 80 Ala Phe Lys 1 <210> 81 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Tissue plasminogen activator (tPA) <400> 81 Gly Gly Ser Gly Gln Arg Gly Arg Lys Ala Leu Glu 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> ADAM10 <400> 82 Pro Arg Tyr Glu Ala Tyr Lys Met Gly Lys 1 5 10 <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ADAM12 <400> 83 Leu Ala Gln Ala Phe 1 5 <210> 84 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ADAM17 <400> 84 Glu His Ala Asp Leu Leu Ala Val Val Ala Lys 1 5 10 <210> 85 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 85 Gly Gly Gly Gly Ser 1 5 <210> 86 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 86 Gly Gly Gly Ser 1 <210> 87 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 87 Gly Ser 1 <210> 88 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 88 Gly Ser Gly Gly Ser 1 5 <210> 89 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 89 Gly Gly Ser Gly 1 <210> 90 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 90 Gly Gly Ser Gly Gly 1 5 <210> 91 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 91 Gly Ser Gly Ser Gly 1 5 <210> 92 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 92 Gly Ser Gly Gly Gly 1 5 <210> 93 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 93 Gly Gly Gly Ser Gly 1 5 <210> 94 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Flexible amino acid linker (can be presented in a repeating manner) <400> 94 Gly Ser Ser Ser Gly 1 5 <210> 95 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamer (tight binder with Kd=2.4 nM) <400> 95 ugccgcuaua augcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 96 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 96 uggcgcuaaa uagcacggaa auaaucgccg uagaaaagca ugucaaagcc g 51 <210> 97 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 97 ugcuaguaua ucgcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 98 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 98 ugccgccaua ucacacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 99 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 99 uuccgcugua uaacacggac uuaaucgccg uaguaaagca ugucaaagcc g 51 <210> 100 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 100 ugucgcucua uugcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 101 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 101 ugcugcuuua ucccacauau uuuuuccccu cauaacaaua uuucuccccc c 51 <210> 102 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (4)..(4) <223> n is a, c, g, or u <220> <221> misc_feature <222> (15)..(15) <223> n is a, c, g, or u <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, or u <220> <221> misc_feature <222> (47)..(47) <223> n is a, c, g, or u <400> 102 ugcngcuaua ucgcncguau uuaaucgccg uagaaaagca ugcnangcc g 51 <210> 103 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 103 ugcaaagaaa acgcacguau uuaaucgccg uaguaaagca ugucaaagcc g 51 <210> 104 <211> 53 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 104 ugcaucacua ucgaaccuau uuaauccacc aaaauaauug caaguccaua cuc 53 <210> 105 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (5)..(6) <223> n is a, c, g, or u <220> <221> misc_feature <222> (17) <223> n is a, c, g, or u <220> <221> misc_feature <222> (48)..(48) <223> n is a, c, g, or u <400> 105 ugccnnaaua acacacnuau auaaucgccg uacaaaauca ugucaaancc g 51 <210> 106 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 106 ugcagcugua uugcacguau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 107 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 107 uuccgauaau cccgcguacu aaaucaccau agucaacaau uuccaaccuc 50 <210> 108 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 108 uccacuauau cacacguauu uaaucgccgu agaaaagcau gucaaagccg 50 <210> 109 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 109 ucccucaacc ucgcuacuau uuaaucgccg uagaaaagca ugucaaagcc u 51 <210> 110 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 110 ugccgcuaua ucacacgaau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 111 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 111 agccccuaga acacacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 112 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 112 ugccaauaua uaacacggaa uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 113 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 113 ugccgcuaua gcgcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 114 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 114 ugcagauaua ugucacucau uaauccccgu auaaaaacau aacuaagcuc 50 <210> 115 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 115 uguagcugua uugcacacau uaaaucgccg uaguaaagca ugucaaagcc g 51 <210> 116 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 116 uaccaauaua ucgccacaca uaaucgccgu agaaaagcau gucaaagccg 50 <210> 117 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 117 ugccgcuaug cccacggaau uuaaucgccg uagaaaaaca ugucaaaguc g 51 <210> 118 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 118 ugccgcuauu uagcacggau uaaaucgccg uagaaaagca ugucaaagcc g 51 <210> 119 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, or u <400> 119 ugccgcuauu uagcacggau uaaaucgccg uagaaaagca ugcnaagcc g 51 <210> 120 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (41)..(41) <223> n is a, c, g, or u <400> 120 uguaguaaua ugacacggau uuaaucgccg uagaaaagca ngucaaagcc u 51 <210> 121 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 121 ugucgccauu acgcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 122 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 122 ugcccccaaa cuacacaaau uuaaucgccg uauaaaagca ugucaaagcc g 51 <210> 123 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 123 ugcacuaucu cacacguacu aaucgccgua uaaaagcaug ucaaagccg 49 <210> 124 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 124 ugucgcaaua auacacuaau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 125 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 125 ugcaacaaua uagcacguau uuaaucgccg uaguaaagca ugucaaagg 49 <210> 126 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 126 cuaccacaaa ucccacauau uuaaucuccc aaucaaaucu uguccauucc c 51 <210> 127 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 127 ugcccuaaac ucacacggau auaaucgccg uagaaaagca ugucaaagcc g 51 <210> 128 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 128 uugucguaug ucacacguau uaaaucgccg uauaaaagca ugucaaagcc g 51 <210> 129 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 129 uuccgcuaua acacacggag aaaaucgccg uaguaaagca ugucaaagcc g 51 <210> 130 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 130 ugccgauaua acgcacggau auaaucgccg uagaaaagca ugucaaagcc g 51 <210> 131 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 131 ugccauuaua cagcacggau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 132 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 132 uccagaaaua ugcacacauu uaaucgccgu agaaaagcau gucaaagccg 50 <210> 133 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 133 uccgcuaaac aacacggaua caaucgccgu agaaaagcau guccaagccg 50 <210> 134 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (46)..(48) <223> n is a, c, g, or u <400> 134 ugcacuaucu cacacguacu aaucgccgua uaaaagcaug ucaaannng 49 <210> 135 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (3)..(3) <223> n is a, c, g, or u <220> <221> misc_feature <222> (6) <223> n is a, c, g, or u <220> <221> misc_feature <222> (8)..(10) <223> n is a, c, g, or u <220> <221> misc_feature <222> (21)..(21) <223> n is a, c, g, or u <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, or u <400> 135 aungcnannn uacacguauu naaucgccgu agaaaagcau gucanagccg 50 <210> 136 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 136 ugcugcuaua uugcaauuuu uuaaacuaag uagaaaacca uguacaaguc g 51 <210> 137 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 137 ugcgccaua uugcacggau uuaaucgccg uagaaaagca ugccaagcc g 51 <210> 138 <211> 52 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 138 ugccguuaua acccacggaa uuuaaccucc guagaaaagc augucaaagc cg 52 <210> 139 <211> 52 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (40)..(40) <223> n is a, c, g, or u <400> 139 ugugaauaua uaucacggau uuaaucgccg uauaaaagcn augucaaagc cg 52 <210> 140 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (10)..(11) <223> n is a, c, g, or u <220> <221> misc_feature <222> (13)..(13) <223> n is a, c, g, or u <400> 140 ugccgauaun nancacggau uuaaucgccg uagaaaagca ugccaagcc g 51 <210> 141 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 141 ugucacuaaa uugcacguau auaaucgccg uaguaagcau gucaaagccg 50 <210> 142 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 142 ugcaaccaua aagcacguaa uaaaucgccg uauauaagca ugucaaagcc g 51 <210> 143 <211> 50 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 143 ugccgcuaua uagcacguau uaaucgccgu aguaaagcau gucaaagccg 50 <210> 144 <211> 52 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 144 ugccgcuaua gcacacggaa uuuaaucgcc guaguaaagc augucaaagc cg 52 <210> 145 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (15)..(15) <223> n is a, c, g, or u <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, or u <400> 145 ugcagguaua uaacncggau uuaaucgccg uagaaaagca ugucnaagcc g 51 <210> 146 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 146 ugcuccuaua acacacggau uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 147 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 147 ugcccguaau ugcacggauu uaaucgccgu agaaaagcau guccaagccg g 51 <210> 148 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <220> <221> misc_feature <222> (12)..(12) <223> n is a, c, g, or u <220> <221> misc_feature <222> (44)..(44) <223> n is a, c, g, or u <400> 148 acucccuaua ungcaacuac auaaucgccg uaaauaagca uguncaagcc g 51 <210> 149 <211> 54 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 149 ugaagcuaga ucacacuaaa uuaaucgccg uagaaaagca ugucaaaaaa gccg 54 <210> 150 <211> 52 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 150 ugacucuuua ucccccguac auuauucacc gaaccaaagc auuaccaucc cc 52 <210> 151 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 151 ugacgcccua acacacguau auaaucgccg uagaaaagca ugucaaagcc g 51 <210> 152 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 152 ugucgcaaaa uagcacguau uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 153 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 153 ugaguguaua auucacguau uuaaucgccg uagaaaagca ugucaaagcc g 51 <210> 154 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 154 ugcuacuaua ucguagguaa cuaaucgccc uacaaacuca cucuaaaacc g 51 <210> 155 <211> 53 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 155 uuacgcuaua ucacacggaa uuuuaaucgc cguagaaaag cauguccaag ccg 53 <210> 156 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 156 cccaucugua cuacaggaau uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 157 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 157 ugcccauaaa uagcacggau uuaaucgccg uagaaaagca ugccaagcc g 51 <210> 158 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 158 ugccgcaaua acauacacau auaaucgccg uagaaaagca ugucaaagcc g 51 <210> 159 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 159 ugcaacuaua ucgcacguau guaaucgccg uagaaaaagc augucaaagc c 51 <210> 160 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 160 uuccgcuaua uagcacggaa uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 161 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 161 uuccgcuaag ucacacgaaa uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 162 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 162 uguagcaaua ucacacguaa uuaaucgccg uauauaagca ugucaaagcc g 51 <210> 163 <211> 51 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 163 ugccguuaua uaucacggau uuaaucgccg uagaaaagca uguccaagcc g 51 <210> 164 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> Anti-EGFR aptamers <400> 164 uaacacauau aucaaguaac uuaucuccuu aguaaccauc uccaagccg 49 <210> 165 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> Anti-EpCAM VH-CH1, SP34 VH, FXa, Basic Coiled <400> 165 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 225 230 235 240 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 245 250 255 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met 260 265 270 Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 275 280 285 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 290 295 300 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr 305 310 315 320 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 325 330 335 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 340 345 350 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 355 360 365 Ile Glu Gly Arg Gly Gly Gly Gly Ser Lys Asn Ala Gln Cys Lys Lys 370 375 380 Lys Leu Gln Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu 385 390 395 400 Gln Ala Leu Lys Lys Lys Leu Ala Gln Gly His His His His His His 405 410 415 <210> 166 <211> 387 <212> PRT <213> Artificial Sequence <220> <223> Substitution [[ID=A]]<A <400> 166 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr ">65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 225 230 235 240 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 245 250 255 Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 260 265 270 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 275 280 285 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 290 295 300 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 305 310 315 320 Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly 325 330 335 Thr Lys Val Glu Ile Lys Gly Gly Pro Leu Gly Val Arg Gly Lys Gly 340 345 350 Gly Gly Ser Glu Asn Ala Gln Cys Glu Lys Glu Leu Gln Ala Leu Glu 355 360 365 Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu 370 375 380 Leu Ala Gln 385 <210> 167 <211> 475 <212> PRT <213> Artificial Sequence <220> <223> Anti-EpCAM VH-CH1, SP34 VH, FXa, IgE CH2 <400> 167 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 225 230 235 240 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 245 250 255 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met 260 265 270 Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 275 280 285 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 290 295 300 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr 305 310 315 320 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 325 330 335 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 340 345 350 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ile Glu Gly Arg 355 360 365 Gly Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly 370 375 380 His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr 385 390 395 400 Pro Gly Thr Ile Gln Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp 405 410 415 Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser 420 425 430 Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg 435 440 445 Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser 450 455 460 Thr Lys Lys Cys Ala His His His His His His 465 470 475 <210> 168 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Anti-EpCAM VL-CL, atezolizumab VL, FXa, IgE CH2 <400> 168 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 225 230 235 240 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 245 250 255 Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 260 265 270 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 275 280 285 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 290 295 300 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 305 310 315 320 Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly 325 330 335 Thr Lys Val Glu Ile Lys Gly Gly Gly Ile Glu Gly Arg Gly Pro Pro 340 345 350 Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro 355 360 365 Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr 370 375 380 Ile Gln Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu 385 390 395 400 Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser 405 410 415 Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr 420 425 430 Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys 435 440 445 Cys Ala 450 <210> 169 <211> 477 <212> PRT[[ID=1s]] <213> Artificial Sequence <220> <223> Anti-EpCAM VH-CH1, SP34 VH, FXa, IgE CH2 <400> 169 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn 20 25 30 Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 225 230 235 240 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu 245 250 255 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met 260 265 270 Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 275 280 285 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 290 295 300 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr 305 310 315 320 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 325 330 335 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 340 345 350 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ile Glu 355 360 365 Gly Arg Gly Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly 370 375 380 Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly 385 390 395 400 Tyr Thr Pro Gly Thr Ile Gln Ile Thr Trp Leu Glu Asp Gly Gln Val 405 410 415 Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu 420 425 430 Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser 435 440 445 Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu 450 455 460 Asp Ser Thr Lys Lys Cys Ala His His His His His His 465 470 475 <210> 170 <211> 578 <212> PRT <213> Artificial Sequence <220> <223> Anti-EpCAM VL-CL, atezolizumab VL, FXa, IgE CH2 <400> 170 Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 225 230 235 240 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 245 250 255 Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr Gln 260 265 270 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 275 280 285 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 290 295 300 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 305 310 315 320 Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala Thr Phe Gly Gln Gly 325 330 335 Thr Lys Val Glu Ile Lys Gly Gly Gly Ile Glu Gly Arg Gly Gly Gly 340 345 350 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 355 360 365 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 370 375 380 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 385 390 395 400 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 405 410 415 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr 420 425 430 Arg Val Val Ser Val Leu Thr Val...

Claims

1. A kit or composition for treating cancer in a patient, the kit or composition comprising: a. A first component, wherein the first component comprises a targeted immune cell binding agent, wherein the targeted immune cell binding agent comprises: i. a first targeting moiety that binds to a tumor antigen expressed by the cancer; ii. a first immune cell binding domain capable of having immune cell binding activity when bound to a second immune cell binding domain, wherein the second immune cell binding domain is not part of the first component, and wherein the first immune cell binding domain is a VH domain or a VL domain; iii. a first complementary binding domain capable of binding an antigen when bound to a second complementary binding domain, wherein (1) the second complementary binding domain is not part of the first component, (2) when the first immune cell binding domain is a VH domain, the first supplementary binding domain is a VL domain, (3) when the first immune cell binding domain is a VL domain, the first supplementary binding domain is a VH domain, and (4) wherein the first complementary binding domain is a binding partner of 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 first immune cell binding domain does not bind to the first complementary domain; iv. a linker connecting the first targeting moiety and the first immune cell binding domain; and v. a linker connecting the first targeting moiety and the first complementary binding domain; and: b. A second component, wherein the second component comprises a targeted immune cell binding agent, wherein the targeted immune cell binding agent comprises: i. a second targeting moiety; ii. a second immune cell binding domain capable of having immune cell binding activity when bound to a first immune cell binding domain, wherein if the first immune cell binding domain is VL, then the second immune cell binding domain is VH, and wherein if the first immune cell binding domain is VH, then the second immune cell binding domain is VL; iii. a second complementary binding domain capable of binding an antigen when bound to said first complementary binding domain, wherein (1) when the second immune cell binding domain is a VH domain, the second complementary binding domain is a VL domain, (2) when the second immune cell binding domain is a VL domain, the second complementary binding domain is a VH domain, and (3) wherein the second complementary binding domain is a binding partner of 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 second immune cell binding domain does not bind to the second complementary domain; iv. a linker connecting the second targeting moiety and the second immune cell binding domain; and v. a linker connecting the second targeting moiety and the second complementary binding domain; The first and second targeting moieties each independently comprise: (i) an antibody or an antigen-binding fragment thereof, (ii) an aptamer, or (iii) 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.

2. The kit or composition of claim 1, wherein the first immune cell binding domain is bound to the first complementary binding domain via a first dimerization domain and a second dimerization domain, wherein: a. the first dimerization domain is connected to the first immune cell binding domain via a first cleavable linker; and b. The second dimerization domain is linked to the first complementary binding domain via a second cleavable linker.

3. The kit or composition of claim 1, wherein the second T cell binding domain is bound to the second complementary binding domain via a first dimerization domain and a second dimerization domain, wherein: a. the first dimerization domain is connected to the second T cell binding domain via a first cleavable linker; and b. The second dimerization domain is linked to the second complementary binding domain via a second cleavable linker.

4. The kit or composition of claim 2, wherein: a. the protease cleavage site of the first cleavable linker and / or the second cleavable linker is cleaved by a protease expressed by the cancer or tumor microenvironment cells; and / or b. The first cleavable linker and the second cleavable linker are 5 to 30 amino acids in length.

5. The kit or composition of claim 4, wherein the first cleavable linker and the second cleavable linker are 8 to 16 amino acids in length.

6. The kit or composition of claim 2, wherein: i) the first dimerization domain and the second dimerization domain are both: a.Leucine zipper; b. an immunoglobulin domain; or cT cell receptor (TCR) domain; and / or ii) the first dimerization domain and the second dimerization domain are both immunoglobulin domains.

7. The kit or composition of claim 1, wherein the first complementary binding domain and the second complementary binding domain, when bound to each other, are capable of binding: a. cancer cells of said cancer; or bT cells, macrophages or natural killer cells.

8. The kit or composition of claim 6, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to T cells when bound to each other.

9. A kit or composition as described in claim 6, wherein the first complementary binding domain and the second complementary binding domain are capable of binding to CD3, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS) when bound to each other.

10. The kit or composition of claim 6, wherein the first complementary binding domain and the second complementary binding domain are capable of binding natural killer cells when associated with each other.

11. The kit or composition of claim 6, wherein the first complementary binding domain and the second complementary binding domain are capable of binding CD16A when bound to each other.

12. The kit or composition of claim 1, wherein: a) the first immune cell binding domain and the second immune cell binding domain are capable of binding to T cells, macrophages or natural killer cells when bound to each other; or b) The first immune cell binding domain and the second immune cell binding domain are capable of binding to T cells when bound to each other.

13. A kit or composition as described in claim 1, wherein the first immune cell binding domain and the second immune cell binding domain are capable of binding to CD3, T cell receptor, programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and 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 co-stimulator (ICOS) when bound to each other.

14. The kit or composition of claim 1, wherein the first targeting moiety and / or the second targeting moiety comprises an antibody or an antigen binding fragment thereof.

15. The kit or composition of claim 1, wherein the first targeting moiety and / or the second targeting moiety comprises an antibody or antigen binding fragment thereof that binds to: α4-integrin; A33; ACVRL 1 / ALK1;ADAM17;ALK;APRIL;BCMA;C242;CA125;Edherin-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;CLDN18.2; 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; epithelial sialoprotein; FAP; FGFR1; FGFR2; FGFR3; FGFR4; fibronectin additional structure 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; TWEAKR; TYRP1; VANGL2; VEGF; VEGF-C; VEGFR-2; or VEGF-R2. .

16. The kit or composition of claim 1, wherein the second targeting moiety binds to an antigen expressed by a cell of the tumor microenvironment.

17. The kit or composition of claim 16, wherein the tumor microenvironment cells are fibroblasts or macrophages.

18. The kit or composition of claim 16 or 17, wherein the antigen expressed by fibroblasts is fibroblast activation protein, or the antigen expressed by macrophages is MAC-1 / CD11b or sideroflexin 3.

19. The kit or composition of claim 1, wherein the first complementary binding domain and the second complementary binding domain, when bound to each other, are capable of binding: a. wherein the first complementary binding domain and the second complementary binding domain are capable of binding to an immune checkpoint molecule, RANK or RANKL, or a cell death inducing antigen when bound to each other; or bT cells, macrophages or natural killer cells.

20. Use of the kit or composition of any one of claims 1 to 19 in the preparation of a medicament for targeting immune cells to a cancer in a patient expressing a tumor antigen that binds both the first targeting moiety and / or the second targeting moiety.

21. Use of the kit or composition of any one of claims 1 to 19 in the preparation of a medicament for targeting immune cells to a cancer in a patient that expresses two tumor antigens, wherein one tumor antigen is bound to a first targeting moiety and one tumor antigen is bound to a second targeting moiety.

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