HK40088417A - Multi-specific antibodies and methods of making and using thereof - Google Patents

Description

Multispecific antibodies and their preparation and use methods

This application is a divisional application of Chinese invention patent application 2018800394014 (Invention title: Multispecific antibody and its preparation and use method; application date: June 22, 2018).

Cross-reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62524557, filed June 25, 2017, which is expressly incorporated herein by reference in its entirety.

Technical Field

This disclosure generally relates to the field of biotherapy technology, and more specifically to the preparation and use of multispecific antibodies.

Background Technology

Cancer cells develop various strategies to evade the immune system. One potential mechanism of immune escape is reduced recognition of cancer cells by the immune system. Defective presentation or absence of cancer-specific antigens leads to immune tolerance and cancer progression. In the presence of effective immune recognition, tumors employ other mechanisms to avoid elimination by the immune system. Immunoreactive tumors generate a suppressive microenvironment that downregulates the immune response. Multiple actors are involved in the formation of the suppressive tumor microenvironment, including tumor cells, regulatory T cells, myeloid-derived suppressor cells, stromal cells, and other cell types. Suppression of the immune response can occur in a cell-contact-dependent manner or in a contact-independent manner by secreting immunosuppressive cytokines from the local environment or by eliminating essential survival factors. Cell contact-dependent inhibition depends on molecules expressed on the cell surface, such as programmed death-ligand 1 (PD-L1), T lymphocyte-associated protein 4 (CTLA-4), etc. [Dunn et al., 2004, Immunity, 21(2): 137-48; Adachi and Tamada, 2015, Cancer Sci., 106(8): 945-50].

As the mechanisms by which tumors evade immune system recognition continue to be better understood, new therapeutic approaches targeting these mechanisms have recently emerged. On March 25, 2011, the U.S. Food and Drug Administration (FDA) approved yrevoy (Bristol-Myers Squibb), an injection of ipilimumab, for the treatment of unresectable or metastatic melanoma. Yrevoy binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressed on activated T cells and blocks the interaction between CTLA-4 and CD80/86 on antigen-presenting cells, thereby blocking negative or inhibitory signals delivered to T cells via CTLA-4, leading to the reactivation of antigen-specific T cells, which in many patients results in tumor eradication. A few years later, in 2014, the FDA approved Keytruda (pembrolizumab, Merck) and Opdivo (nivolumab, Bristol-Myers Squibb) for the treatment of advanced melanoma. These monoclonal antibodies bind to PD-1 expressed on activated and/or depleted T cells and block the interaction between PD-1 and PD-L1 expressed on tumors, thereby eliminating the inhibitory signal that enters T cells via PD-1, leading to the reactivation of antigen-specific T cells, which in many patients again leads to tumor eradication. Since then, additional clinical trials have been conducted comparing the single monoclonal antibody Yervoy with the combination of the monoclonal antibodies Yervoy and Opdivo in the treatment of advanced melanoma, which showed improvements in overall survival and progression-free survival in patients treated with the antibody combination. (Hodi et al., 2016, Lancet Oncol. 17(11):1558-1568; Hellman et al., 2018, Cancer Cell 33(5):853-861). However, since many clinical trials have demonstrated the significant benefits of treating cancer patients with monoclonal antibodies specific to one or more immune checkpoint molecules, data have shown that only those patients with a high mutational burden producing novel T-cell epitopes recognized by antigen-specific T cells have shown clinical responses (Snyder et al., 2014, NEJM371: 2189-2199). Those patients with low tumor mutational burden have largely not shown objective clinical responses (Snyder et al., 2014, NEJM371: 2189-2199; Hellman et al., 2018, Cancer Cell 33(5): 853-861).

In recent years, other teams have developed alternative methods to activate T cells without the need for the presentation of novel epitopes by antigen-presenting cells. One example is the development of bispecific antibodies in which the binding domain of an antibody specific to tumor-associated antigens such as CD19 is linked to the binding domain of an antibody specific to CD3 on T cells, thereby creating a bispecific T cell binder or binding molecule. In 2014, the FDA approved a bispecific antibody called blinatumomab for the treatment of proto-B-cell acute lymphoblastic leukemia. blinatumomab links a CD19-specific scFv expressed on leukemia cells to a CD3-specific scFv expressed on T cells (Bejnijamin and Stein 2016, Ther Adv Hematol 7(3): 142-146). However, despite initial response rates >50% in patients with relapsed or refractory acute lymphoblastic leukemia (ALL), many patients develop resistance or relapse to blinatumomab after successful treatment. Evidence suggests that resistance to blinatumomab or relapse following blinatumomab treatment is attributable to the expression of immune checkpoint inhibitory molecules, such as PD-L1, expressed on tumor cells, which drive inhibitory signaling via PD-1 expression on activated T cells (Feucht et al., 2016, Oncotarget7 (47): 76902-76919). In a case study of patients resistant to blinatumomab, a second round of blinatumomab treatment was administered, but with the addition of Pembrol izumab (Keytruda, Merck), a monoclonal antibody that is PD-1 specific and blocks the interaction between PD-1 expressed on T cells and PD-L1 expressed on tumor cells. This resulted in a significant response of tumor cells in the bone marrow of this patient, with the percentage decreasing from 45% to less than 5% (Feucht et al., 2016, Oncotarget 7(47): 76902-76919). These results suggest that combining a bispecific biting molecule with one or more monoclonal antibodies can significantly increase clinical activity compared to either agent alone.

Summary of the Invention

This disclosure particularly provides a tetraspecific antibody monomer, an antibody containing a tetraspecific monomer, an antigen-binding fragment thereof, a multispecific antibody, an immunoconjugate containing the disclosed antibody, a method for preparing the disclosed monomer, antigen-binding fragment and antibody, and a method for treating cancer using the disclosed molecule.

On one hand, this application provides a four-specific antibody monomer. In one embodiment, the four-specific antibody monomer having an N-terminus and a C-terminus sequentially comprises, from the N-terminus to the C-terminus, a first scFv domain, a Fab domain, an Fc domain, a second scFv domain, and a third scFv domain at the C-terminus. Each of the first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain has binding specificity against a different antigen.

In one embodiment, the antigen includes a tumor antigen, an immune signaling antigen, or a combination thereof. In one embodiment, the first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each have binding specificity against a tumor antigen or an immune signaling antigen. In one embodiment, the first scFv domain has binding specificity against a tumor antigen. In one embodiment, the first scFv domain has binding specificity against an immune signaling antigen. In one embodiment, the Fab domain has binding specificity against a tumor antigen. In one embodiment, the Fab domain has binding specificity against an immune signaling antigen. In one embodiment, the second scFv domain has binding specificity against a tumor antigen. In one embodiment, the second scFv domain has binding specificity against an immune signaling antigen. In one embodiment, the third scFv domain has binding specificity against a tumor antigen. In one embodiment, the third scFv domain has binding specificity against a tumor antigen.

In one embodiment, the four-specific monomer includes an scFv domain, a Fab domain, a second scFv domain, and a third scFv domain, each independently possessing binding specificity against an antigen selected from: ROR1, PD-L1, CD3, CD28, 4-1BB, CEA, HER2, EGFR VIII, EGFR, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypimay-3, gpA33, GD2, TROP2, NKG2D, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73, and CD39. In one embodiment, the scFv domain, Fab domain, second scFv domain, and third scFv domain each independently possess binding specificity for tumor-specific antigens, including but not limited to ROR1, CEA, HER2, EGFR, EGFR VIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypimay-3, gpA33, GD2, TROP2, BCMA, CD3, CD19, CD20, CD33, CD123, CD22, CD30, or immune checkpoint modulators, including but not limited to PD-L1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, CD39, etc. In one embodiment, a set of scFv domains can specifically bind to immune checkpoint modulators or tumor antigens. In one implementation, the CD3-specific scFv can be located at the C or N end of the heavy or light chain.

In one embodiment, the first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each independently possess binding specificity against an antigen selected from CD3, EGRF VIII, PD-L1, and 4-1BB. In one embodiment, the first scFv domain has binding specificity against CD3. In one embodiment, the Fab domain has binding specificity against EGRF VIII. In one embodiment, the second scFv domain has binding specificity against PD-L1. In one embodiment, the third scFv domain has binding specificity against 4-1BB. In one embodiment, the first scFv domain has binding specificity against CD3, the Fab domain has binding specificity against EGRF VIII, the second scFv domain has binding specificity against PD-L1, and the third scFv domain has binding specificity against 4-1BB.

The Fc domain can be humanized. In one embodiment, the Fc domain is human IgG1 Fc.

The scFv domain may include a linker that connects the scFv domain to the heavy or light chain of the antibody. In one embodiment, the linker may include more than 10 amino acids. In one embodiment, the linker may include more than 15 amino acids in length. In one embodiment, the linker may include fewer than 20 amino acids.

In one implementation, the connector may include a gly-gly-gly-gly-ser(G4S)n connector, where n is an integer from 1 to 20. For example, n may be 2, 4, or 6. In one implementation, the first scFv structural domain, the second scFv structural domain, or the third scFv structural domain may include a gly-gly-gly-gly-ser(G4S)n connector, where n is 2 or 4.

In one embodiment, this application provides a four-specific antibody monomer having an amino acid sequence having a percentage homology with the following amino acid sequences: SEQ ID NO. 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60. The percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

This application also provides antigen-binding fragments. In one embodiment, this application provides an scFv domain. In one embodiment, the scFv domain comprises amino acid sequences having a percentage homology with the following amino acid sequences: SEQ ID NO. 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60, wherein the percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, this application provides a Fab domain. In one embodiment, the fab domain comprises an amino acid sequence having a percentage homology with the following amino acid sequences: SEQ ID NO. 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60, wherein the percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%. The antigen-binding fragments disclosed herein can be used to construct tetraspecific antibody monomers or multispecific antibodies.

On one hand, this application provides multispecific antibodies. In one embodiment, the multispecific antibody comprises a tetraspecific antibody monomer. In one embodiment, the multispecific antibody comprises two tetraspecific antibody monomers disclosed herein. Since each tetraspecific antibody monomer has four antigen-binding domains, the disclosed multispecific antibody may comprise eight antigen-binding domains. In one embodiment, the antigen-binding domains in such a multispecific antibody each independently have binding specificity against different antigens, thereby providing an eight-specific antibody. In one embodiment, the multispecific antibody is a five-specific antibody. In one embodiment, the multispecific antibody is a five-specific antibody. In one embodiment, the multispecific antibody is a five-specific antibody and a six-specific antibody. In one embodiment, the multispecific antibody is a five-specific antibody and a seven-specific antibody.

In one embodiment, the multispecific antibody comprises a dimer of a tetraspecific antibody monomer, thereby providing a tetraspecific antibody. In one embodiment, this application provides isolated, purified, or non-naturally occurring multispecific antibodies. In one embodiment, this application provides a tetraspecific antibody having an amino acid sequence having a percentage homology with SEQ ID NO. 66 and 68. The percentage homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

This application also provides isolated nucleic acid sequences encoding tetraspecific antibody monomers, multispecific antibodies, or antigen-binding fragments thereof. In one embodiment, the nucleic acid encodes an amino acid sequence having a percentage homology with a tetraspecific antibody monomer having the following amino acid sequences: SEQ ID NO. 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and 59. The percentage of homology is not less than 70%, 80%, 90%, 95%, 98%, or 99%.

This application also provides an expression vector comprising the nucleic acid sequences disclosed herein and a host cell. In one embodiment, the host cell comprises the expression vector. The host cell may be a prokaryotic cell or a eukaryotic cell.

This application also provides immunoconjugates. In one embodiment, the immunoconjugate includes a cytotoxic agent or imaging agent linked to a multispecific antibody disclosed herein via a linker.

The connector may be cuttable or non-cuttable. The connector may include covalent bonds, such as ester bonds, ether bonds, amide bonds, disulfide bonds, imide bonds, sulfone bonds, phosphate bonds, phosphate ester bonds, peptide bonds, or combinations thereof. In one embodiment, the connector comprises a hydrophobic poly(ethylene glycol) connector.

Cytotoxic agents may include chemotherapeutic agents, growth inhibitors, cytotoxic agents derived from the calicheamicin class, antimitotic agents, toxins, radioisotopes, therapeutic agents, or combinations thereof. In one embodiment, the cytotoxic agent is selected from calicheamicin, ozogamicin, monomethylaurestatin E, emtansine, derivatives thereof, or combinations thereof.

The imaging agent can be any compound used for imaging purposes. In one embodiment, the imaging agent can be a radionuclide, a fluorescent agent, a quantum dot, or a combination thereof.

This application also provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a tetraspecific antibody monomer disclosed herein. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a multispecific antibody disclosed herein. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an antigen-binding fragment disclosed herein. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an immunoconjugate disclosed herein.

In one embodiment, the pharmaceutical composition further includes a therapeutic agent. Examples of therapeutic agents include, but are not limited to, radioisotopes, radionuclides, toxins, chemotherapeutic agents, or combinations thereof. In one embodiment, the therapeutic agent comprises an antibody, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent comprises an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof. In one embodiment, the therapeutic agent comprises a checkpoint inhibitor. In one embodiment, the therapeutic agent comprises inhibitors of: PD1, PDL1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, derivatives thereof, or combinations thereof.

On the other hand, this application provides methods for preparing tetraspecific antibody monomers, multispecific antibodies, their antigen-binding fragments, and their immunoconjugates.

In one embodiment, the method includes the steps of culturing host cells containing the nucleic acid sequences disclosed herein to express a DNA sequence encoding an antibody and purifying the antibody. In one embodiment, the antibody is a trispecific antibody.

On the other hand, this application provides a method for using a tetraspecific antibody monomer, a multispecific antibody, its antigen-binding fragment, and its immunoconjugates for cancer treatment. In one embodiment, the method includes administering a tetraspecific antibody monomer, a multispecific antibody, its antigen-binding fragment, its immunoconjugate, or a pharmaceutical composition thereof to a subject in need of such treatment. In one embodiment, the method includes administering an effective amount of the tetraspecific antibody to the subject.

In one embodiment, the method includes directly injecting an effective amount of a multispecific monomer, a multispecific antibody, an immune conjugate, or its antigen-binding fragment directly into the tumor site.

It can prevent or treat a variety of cancers. In one implementation, the cancer may have cells expressing ROR1, CEA, HER2, EGFR, EGFR VIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, phosphatidylinositol proteoglycan-3, gpA33, GD2, TROP2, NKG2D, BCMA, CD19, CD20, CD33, CD123, CD22, or CD30. Examples of cancers include, but are not limited to, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain tumors, gliomas, neuroblastomas, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma.

In one embodiment, the method may further include co-administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent includes an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent includes an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof. In one embodiment, the therapeutic agent may include a checkpoint inhibitor. In one embodiment, the therapeutic agent includes inhibitors of PD1, PD-L1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, derivatives, or combinations thereof.

In one implementation, the therapeutic agents may include capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, vorazole, and thiamethoxam. Stanozolol, erlotinib, lafatinib, dasatinib, gefitinib, osimertinib, van der tanib, afatinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, NaB-paratacin, everolimus, tesimolimus, dabrafenib, vemorafenib, trametinib, vincafolic acid, apatinib, crizotinib, perifosine, olaparib, bortezomib, tofatinib, trastuzumab, and their derivatives or combinations thereof.

The subject can be a human being. In one embodiment, the subject may have cancer. This application also provides a solution containing an effective concentration of the multispecific antibody, monomer, or immunoconjugate disclosed herein. In one embodiment, the solution is the plasma of the subject.

The objects and advantages of the invention will become apparent from the following detailed description of exemplary embodiments of the invention, taken in conjunction with the accompanying drawings. Other embodiments will be apparent to those skilled in the art from the following detailed description, wherein the embodiments are described by way of illustrating the expected best mode. It will be appreciated that other and different embodiments are possible, and that several details of the embodiments can be modified in various obvious ways without departing from their spirit and scope. Therefore, the drawings and detailed description should be considered illustrative in nature rather than restrictive.

Attached Figure Description

The foregoing and other features of the invention will become more fully apparent from the accompanying drawings, the following description, and the appended claims. It should be understood that these drawings depict only a few embodiments of the arrangements according to this disclosure and are therefore not to be considered as limiting its scope. The disclosure will be described with additional features and details using the drawings, in which:

Figure 1 is a schematic diagram of a four-specific antibody having domains 1-4 as antigen-binding domains. The CD3×EGFRvIII×PD-L1×4-1BB four-specific antibody is shown as an example according to one embodiment.

Figure 2 depicts the experimental results showing the cytotoxicity of redirected PBMCs (peripheral blood mononuclear cells) against the EGFRvIII-transfected astrocytoma cell line U87. The tumor lysis activity of the four specific antibodies used is listed in Table 1.

Figure 3 depicts the experimental results showing the cytotoxicity of redirected PBMCs (peripheral blood mononuclear cells) against the Kasumi-2 acute lymphoblastic leukemia cell line.

Figure 4 depicts the experimental results showing the cytotoxicity of redirected PBMCs (peripheral blood mononuclear cells) against the EGFRvIII-transfected astrocytoma cell line U87. The functional activity of different 4-1BB domains and the functional effects of PD-L1 and the 4-1BB domains are shown.

Figure 5 shows the experimental results of FACS analysis of four specific antibodies against human ROR1-transfected CHO cells according to some implementation schemes.

Figure 6 is a depiction of experimental results of FACS analysis of four specific antibodies against human 4-1BB transfected CHO cells according to some implementation schemes.

Figure 7 is a depiction of experimental results of FACS analysis of four specific antibodies in combination with human PD-L1 transfected CHO cells according to some implementation schemes.

Figure 8 illustrates the experimental results of a redirected T cell cytotoxicity (RTCC) assay mediated by a four-specific antibody having a binding domain 323H7 that specifically targets the Ig domain of ROR1, using peripheral blood mononuclear cells as the effector and the B-ALL cell line Kasumi2 as the target, according to some embodiments.

Figure 8 illustrates the experimental results of a redirected T cell cytotoxicity (RTCC) assay mediated by a four-specific antibody having a binding domain 323H7 that specifically targets the Ig domain of ROR1, using peripheral blood mononuclear cells as the effector and the B-ALL cell line Kasumi2 as the target, according to some embodiments.

Figure 9 illustrates the experimental results of a retargeted T cell cytotoxicity (RTCC) assay mediated by a quadruple specific antibody with a binding domain 323H7 that specifically targets the Ig domain of ROR1, using CD8+ and CD45RO+ memory T cells as effectors and the B-ALL cell line Kasumi2 as the target, according to some implementation schemes.

Figure 10 illustrates the experimental results of a retargeted T cell cytotoxicity (RTCC) assay mediated by a quadruple specific antibody having a binding domain 323H7 that specifically targets the Ig domain of ROR1, using CD8+, CD45RA+ naïve T cells as effectors and the B-ALL cell line Kasumi2 as a target, according to some implementation schemes.

Figure 11 illustrates the experimental results of a redirected T cell cytotoxicity (RTCC) assay mediated by a four-specific antibody having a binding domain 338H4 that specifically targets the coiled domain of ROR1, using peripheral blood mononuclear cells as the effector and the B-ALL cell line Kasumi2 as the target, according to some embodiments.

Figure 12 illustrates the experimental results of a retargeted T cell cytotoxicity (RTCC) assay mediated by a quadruple specific antibody with a binding domain 338H4 that specifically targets the coiled domain of ROR1, using CD8+, CD45RO+ memory T cells as effectors and the B-ALL cell line Kasumi2 as a target, according to some implementation schemes.

Figure 13 illustrates the experimental results of a retargeted T cell cytotoxicity (RTCC) assay mediated by a four-specific antibody with a specific binding domain 338H4 targeting the coiled domain of ROR1, using CD8+, CD45RA+ naïve T cells as effectors and the B-ALL cell line Kasumi2 as the target, according to some implementation schemes.

Detailed Implementation

In the following detailed description, reference is made to the accompanying drawings, which form a part of this document. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that these aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly contemplated herein.

This disclosure provides, in particular, isolated antibodies, methods for preparing said antibodies, tetraspecific or multispecific molecules, antibody-drug conjugates and/or immunoconjugates composed of said antibodies or antigen-binding fragments, pharmaceutical compositions containing said antibodies, tetraspecific or multispecific molecules, antibody-drug conjugates and/or immunoconjugates, methods for their preparation, and methods for treating cancer using the disclosed molecules or compositions.

The term "antibody" is used in the broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments (e.g., Fab, F(ab') ₂ , and Fv), provided they exhibit the desired biological activity. In some embodiments, antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, tetraspecific antibodies or bispecific antibodies, humanized antibodies, human antibodies, and humanized antibodies and their active fragments. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab') ₂ , scFv, and Fv fragments, as well as products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind to antigens. Immunoglobulins can be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. In one embodiment, an antibody can be a complete antibody and any antigen-binding fragment derived from that complete antibody. A typical antibody is a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The VH and VL regions can be further subdivided into a hypervariable complementarity-determining region (CDR) domain and a more conserved region called a framework region (FR). Each variable region (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with antigens exist within the variable regions of both the light and heavy chains.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that individual antibodies comprising that group are identical except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies exhibit high specificity against a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized from hybridoma cultures, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates that the properties of the obtained antibody derive from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by a hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567).

Monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular class or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another class or belonging to another antibody class or subclass, as well as fragments of those antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 [1984]).

Monoclonal antibodies can be produced using various methods, including mouse hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9: 15-22 (2002) review) or molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotech NO. 1. 28: 453-7 (2011)). In this invention, antibodies are produced by immunizing rabbits with human PD-L1 protein and cells transiently expressing human PD-L1 on their cell surface. Rabbits are known to produce antibodies with high affinity, diversity, and specificity (Weber et al. Exp. Mol. Med. 49: E305). B cells from immunized animals are cultured in vitro and screened for the production of anti-PD-L1 antibodies. Antibody variant genes are isolated using recombinant DNA technology, the resulting antibodies are recombinantly expressed, and further screened for desired characteristics, such as the ability to inhibit PD-L1 binding to PD-1, the ability to bind to non-human primate PD-L1, and the ability to enhance human T cell activation. The general approach to antibody discovery is similar to that described by Seeber et al. in PLOS One.9:E86184 (2014).

The term "antigen or epitope binding portion or fragment" refers to an antibody fragment capable of binding an antigen (in this case, PD-L1). These fragments can possess both the antigen-binding function and additional functions of a complete antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) or Fab fragments, wherein the single-chain Fv fragment (scFv) consists of the VL and VH domains of a single arm of an antibody linked to a single polypeptide chain via a synthetic linker, and the Fab fragment is a monovalent fragment consisting of the VL, constant light chain (CL), VH, and constant heavy chain 1 (CH1) domains. Antibody fragments can be even smaller subfractions and can consist of domains as small as a single CDR domain, particularly the CDR3 region from the VL and/or VH domains (see, for example, Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are generated using conventional methods known to those skilled in the art. The utility of antibody fragments can be screened using the same techniques as for complete antibodies.

The “antigen or epitope binding fragment” can be derived from the antibody disclosed herein by many techniques known in the art. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. The appropriate fraction containing the Fab fragment can then be collected and concentrated by membrane filtration or the like. For a further description of general techniques for isolating active antibody fragments, see, for example, Khaw, B.A. et al. J. nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

Antibody digestion with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab') ₂ fragment, which has two antigen-binding sites and is still capable of cross-linking the antigen.

Fab fragments may contain a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that they have residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH in this paper refers to Fab', where the cysteine residues in the constant domain have a free thiol group. F(ab')2 antibody fragments are initially generated as Fab' fragment pairs with a hinge cysteine residue between them. Furthermore, chemical coupling of antibody fragments is also known.

"Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly non-covalently bound heavy chain and a light chain variable domain. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind antigens, although with lower affinity than the entire binding site.

Based on the amino acid sequence of their constant structural domains, the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ).

Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; and IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

"Humanized antibody" refers to a class of engineered antibodies whose CDR is derived from a non-human donor immunoglobulin, and whose remaining immunoglobulin-derived portion of the molecule is derived from one (or more) human immunoglobulins. In some embodiments, the framework support residues may be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, for example, Queen et al., Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson et al., Bio/TechNO.logy, 9: 421 (1991)).

As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and are defined as referring to a biomolecule composed of amino acids linked together by peptide bonds.

As used herein, the terms “a,” “an,” and “the” are defined to mean “one or more” and include plural forms, unless the context is inappropriate.

"Isolated" means a biomolecule that does not contain at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, "isolated" refers to a polypeptide that has been identified and isolated and/or recovered from the cells or cell cultures in which it is expressed. Typically, isolated polypeptides can be prepared by at least one purification step. "Isolated antibody" refers to an antibody that substantially does not contain other antibodies with different antigen specificities.

"Recombination" refers to the use of recombinant nucleic acid technology to produce antibodies in foreign host cells.

The term "antigen" refers to an entity or segment thereof that can induce an immune response in an organism, particularly animals, and more particularly mammals, including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

As used herein, the term "immunogenicity" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent and contributes to an immune response in a human or animal. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells in response to the immunogenic composition of this disclosure to alleviate or reduce the condition to be treated.

"Specific binding," or "specifically binding to," or "specifically targeting," refers to binding to a specific antigen or epitope that is significantly different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule to a control molecule, typically a molecule with a similar structure that does not have binding activity. For instance, specific binding can be determined by competition with a control molecule that resembles the target.

Specific binding to a particular antigen or epitope can be characterized, for example, by an antibody's KD relative to the antigen or epitope being at least about ^10⁻⁴ M, at least about ^10⁻⁵ M, at least about ^10⁻⁶ M, at least about ^10⁻⁶ M, at least about ^10⁻⁷ M, at least about ^10⁻⁸ M, at least about ^10⁻⁹ M, or at least about ^10⁻¹⁰ M, at least about ^10⁻¹¹ M, at least about ^10⁻¹² M, or higher, where KD refers to the dissociation rate of a specific antibody-antigen interaction. Typically, antibodies that specifically bind to antigens can have a KD of 20⁻, 50⁻, 100⁻, 500⁻, 1000⁻, 5000⁻, 1000⁻, or more times relative to the antigen or epitope compared to a control.

Similarly, specific binding to a particular antigen or epitope can be manifested, for example, by the antibody’s KA or Ka relative to the control for the epitope being at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times, where KA or Ka refers to the binding rate of the specific antibody-antigen interaction.

The “homology” between two sequences is determined by sequence identity. If the two sequences being compared are of different lengths, sequence identity preferably refers to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues in the longer sequence. Sequence identity can be routinely determined using computer programs. Deviations that occur in comparisons between a given sequence and the sequences described above in this disclosure can be caused by, for example, additions, deletions, substitutions, insertions, or recombinations.

On one hand, this application provides a tetraspecific antibody monomer, its antigen-binding fragment, and a multispecific antibody. In one embodiment, this application provides a tetraspecific antibody.

In one embodiment, this disclosure provides a tetraspecific antibody having binding specificity against four different antigen targets. In one embodiment, the antigen targets are tumor-specific antigens, T-cell receptor CD3 components, or immune checkpoint molecules. The tetraspecific antibody can bind directly to the body's endogenous T cells to kill tumor cells, independent of MHC presentation of tumor antigens to antigen-specific T-cell receptors. In some embodiments, the immune checkpoint regulatory component of the tetraspecific antibody can overcome the immunosuppressive tumor microenvironment to fully activate depleted T cells within the tumor microenvironment.

Tetraspecific antibodies possess the unique property of directly binding to T cells while simultaneously modulating immune checkpoints or inhibiting Tregs or other suppressive immune cells, or targeting tumors with components targeting tumor antigens. This could be beneficial for patients unsuitable for occlusive or CAR-T therapy. In one implementation, tetraspecific antibodies can demonstrate clinical benefit in solid tumors, where similar techniques or CAR-T therapy also show clinical benefit due to limitations imposed by the suppressive tumor microenvironment.

In one embodiment, this application provides engineered antibodies or "quadrispecific" antibodies having four different binding domains. One binding domain is specific for CD3 on T cells, the second binding domain is specific for tumor-associated antigens, including but not limited to ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypimay-3, gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, and the third and fourth binding domains are specific for two different immune checkpoint modulators, such as PDL1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, Light, HVEM, CD73, CD39, etc.

Examples of the four-specific molecules disclosed in this article (Figure 1) target human ROR1 (SEQ ID 33-48), human CD19 (SEQ ID 53-56), or EGFR vIII (SEQ ID 49-52) as tumor-associated antigens. Each of these targeted four-specific proteins also carries an anti-human PD-L1 (SEQ ID 9-16), an anti-human 4-1BB (SEQ ID 21-32), and an anti-human CD3 binding domain (SEQ ID 1-8). These binding domains are converted to scFv and VLVH for placement in the N-terminal domain 1 (D1) of the peptide (Figure 1), or scFv and VHVL for placement in the C-terminal domains 3 (D3) and 4 (D4) of the peptide.

In one embodiment, the scFv molecule described herein contains a 20-amino acid flexible gly-gly-gly-gly-ser (G4S)×4 linker that operatively links VH and VL regardless of V region orientation (LH or HL). The remaining position in the four-specific protein, domain 2 (D2), consists of the IgG1 heavy chain VH-CH1-hinge-CH2-CH3 and its corresponding light chain VL-CL, which can be either a κ or λ chain. D1 and D2 are genetically linked via a 10-amino acid (G4S)×2 linker, and D2, D3, and D4 similarly produce a continuous ~150 kDa heavy chain monomeric peptide. When co-transfected with a suitable light chain, the final symmetric four-specific peptide can be purified by IgG1Fc (protein A/protein G) and analyzed to assess functional activity. Pre-constructed heavy and light chain gene “boxes” allow the use of restriction enzyme sites (HindIII/NheI for the heavy chain and HindIII/BsiWI for the light chain) or “unrestricted cloning” such as Gibson assembly (SGI-DNA, La Jolla, California), infusion (Takara Bio, USA) or NEBuilder (NEB, Ipswich, Massachusetts) to clone the V region, the latter of which is used here.

The four-specific protein was generated through a process that included designing the complete molecule, synthesizing and cloning the nucleotide sequence of each domain, expressing the final product in mammalian cells, and purifying the final product. The nucleotide sequence was assembled using the Geneous 10.2.3 software package (Biomaterials, Auckland, New Zealand) and broken down into its constituent domains for gene synthesis (Genewiz, South Plainfield, New Jersey).

In this embodiment, SI-35E18 (SEQ ID 65 and 67) was divided into its component domains, with anti-4-1BBscFv VLVH occupying D1, anti-human PD-L1 clone PL230C6 occupying D2 (Fab position), anti-human ROR1 Ig domain-specific clone 323H7 VHVL scFv occupying D3, and anti-human CD3 scFv VHVL occupying the C-terminus D4. Using a NEBuilder Web-based tool, 5' and 3' nucleotides were attached to each domain according to its position in the larger protein, such that each domain overlapped with its flanking domains by 20-30 nucleotides. These nucleotides guided site-specific recombination, thereby genetically fusing each domain in a single gene assembly step. Due to the large number of homologous regions in the four-specific nucleotide sequences, N-terminal domains 1 and 2 were assembled separately from C-terminal domains D3 and D4. The N- and C-terminal fragments were then assembled together in a second NEBuilder reaction.

A small aliquot of the sample was transformed into *E. coli* DH10b (Invitrogen, Carlsbad, California) and plated on TB + carbenicillin 100 μg/ml plates (TekNO.va, Hollister, California) and incubated overnight at 37°C. Colonies were selected, and 2 ml of the overnight culture was inoculated into TB + carbenicillin. DNA was prepared from the overnight culture (Thermo-Fisher, Carlsbad, California) and then sequenced using sequencing primers flanking each domain (Sigma, St. Louis, Missouri) (Genewiz, South Plainfield, New Jersey). In some embodiments, the DNA sequences were assembled and analyzed in Genewiz.

On the other hand, this application provides pharmaceutical compositions comprising multispecific antibody monomers, multispecific antibodies, antigen-binding fragments and their immunoconjugates, as well as methods for treating cancer using the disclosed antibodies or pharmaceutical compositions.

The advantages of using the disclosed tetraspecific antibody monomers, multispecific antibodies, or compositions for therapeutic purposes, compared to any existing therapy, include in particular: 1) the inclusion of the IgG Fc domain can confer a longer half-life in serum compared to bispecific molecule-specific molecules; 2) the inclusion of two binding domains specific to immune checkpoint modulators, which can inhibit inhibitory pathways and simultaneously participate in co-stimulatory pathways; and 3) crosslinking CD3 on T cells with tumor-associated antigens, thereby “redirecting” T cells to kill tumors without removing T cells from the patient and genetically modifying them to be specific to tumor cells, as is done for chimeric antigen receptor T cells (CAR-T), before reintroducing them into the patient.

The formulation of pharmaceutical compositions can be performed according to standard methods known to those skilled in the art.

In one embodiment, the antibodies and monomers according to this disclosure can be prepared in a physiologically acceptable formulation and can include pharmaceutically acceptable carriers, diluents, and/or excipients using known techniques. For example, the antibodies according to this disclosure may include any functionally equivalent antibody or its functional portion; in particular, monoclonal antibodies including any functionally equivalent antibody or its functional portion may be combined with pharmaceutically acceptable carriers, diluents, and/or excipients to form a therapeutic composition. The formulation of pharmaceutical compositions according to this disclosure can be performed according to standard methods known to those skilled in the art.

Regarding formulations for administration of suitable compositions to subjects, such as human patients requiring treatment, the antibodies disclosed herein may be mixed or combined with pharmaceutically acceptable carriers known in the art, depending on the chosen route of administration. There are no particular limitations on the administration methods of the antibodies disclosed herein, and suitable routes of administration and the selection of suitable compositions are known in the art and require no extensive experimentation.

Suitable drug carriers, diluents and/or excipients are well known in the art, including, for example, phosphate-buffered saline solutions, water, and emulsions such as oil/water emulsions.

"Pharmaceutical acceptable" means that compounds, materials, compositions, and dosage forms are suitable for use in human or animal tissues without excessive toxicity, irritation, or other problems or complications, within the limits of reasonable medical judgment, and in proportion to a reasonable benefit/risk ratio.

In one embodiment, the pharmaceutical composition may include a protein carrier, such as, for example, serum albumin or immunoglobulin, particularly of human origin. Other bioactive agents may be present in the pharmaceutical compositions of this disclosure, depending on the intended use. In one embodiment, the protein pharmaceutically active substance may be present in amounts from 1 ng to 10 mg per dose. Typically, the administration regimen should be in the range of 0.1 μg to 10 mg of the antibody according to this disclosure, particularly in the range of 1.0 μg to 10 mg, and more particularly in the range between 1.0 μg and 100 μg, and all independent values falling within these ranges are also part of this disclosure. If administration is by continuous infusion, a more suitable dose may be in the range of 0.01 μg to 10 mg units/kg body weight/hour, and all independent values falling within these ranges are also part of this disclosure.

The composition can be administered to a subject in a suitable pharmaceutically effective dose in solid, liquid, or aerosol form. Examples of solid compositions include pills, creams, and implantable dosing units. Pills can be administered orally. Therapeutic creams can be applied topically. Implantable dosing units can be applied topically, such as at a tumor site, or can be implanted for systemic release of the therapeutic composition, such as subcutaneous administration. Examples of liquid compositions include formulations suitable for intramuscular, subcutaneous, intravenous, and intra-arterial injection, as well as formulations for topical and intraocular application. Examples of aerosol formulations include inhaled formulations for administration to the lungs.

As is well known to those skilled in the art, the dosage of a composition will depend on various factors, such as the condition being treated, the specific composition used, and other clinical factors, such as the patient’s weight, size, sex and general health status, body surface area, the specific compound or composition applied, other drugs administered concurrently, and the route of administration.

The term "therapeutic effective amount" or "effective amount" refers to the amount of antibody that, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect (e.g., improvement of the subject's disease) in said human or animal. An effective amount is readily determined by those skilled in the art using conventional methods. When the disease is cancer, an effective amount of the drug may inhibit (e.g., to a certain extent slow, suppress, or stop) one or more of the following exemplary features, including, but not limited to, cancer cell growth, cancer cell proliferation, cancer cell motility, cancer cell infiltration into surrounding organs, tumor metastasis, and tumor growth. When the disease is cancer, an effective amount of the drug may alternatively, when administered to a subject, perform one or more of the following: slow or stop tumor growth, reduce tumor size (e.g., volume or mass), alleviate one or more cancer-related symptoms to a certain extent, prolong progression-free survival, result in an objective response (including, for example, a partial or complete response), and increase overall survival. It is cytoseptic and/or cytotoxic to the extent that the drug can prevent the growth and/or kill existing cancer cells.

Those skilled in the art can determine the effective amount or concentration of the disclosed antibody for the effective treatment of conditions such as cancer. Other parameters, such as the proportions of the various components in the pharmaceutical composition, the dosage and frequency of administration, can be obtained by those skilled in the art without extensive experimentation. For example, a suitable solution for injection may contain, but is not limited to, about 1 to about 20, about 1 to about 10 mg of antibody/ml. Exemplary dosages may be, but are not limited to, about 0.1 to about 20, about 1 to about 5 mg/kg body weight. Exemplary administration frequencies may be, but are not limited to, once daily or three times weekly.

The composition can be administered via standard routes of administration. Typically, the composition can be administered topically, orally, rectally, nasally, intradermally, intraperitoneally, or parenterally (e.g., intravenously, subcutaneously, or intramuscularly). Alternatively, the composition can be incorporated into a sustained-release matrix such as a biodegradable polymer, which can be implanted near the site of delivery, such as a tumor site. The method includes administering a single dose, administering repeated doses at predetermined time intervals, and continuing administration for a predetermined duration.

While many forms of administration are possible, an exemplary form of administration may be a solution for injection, particularly a solution for intravenous or intra-arterial injection. Typically, a suitable pharmaceutical composition for injection may include a pharmaceutically suitable carrier or excipient, such as, but not limited to, buffers, surfactants, or stabilizers. Examples of buffers may include, but are not limited to, acetate, phosphate, or citrate buffers. Examples of surfactants may include, but are not limited to, polysorbate. Examples of stabilizers may include, but are not limited to, human albumin.

In one embodiment, administration may be parenteral, such as intravenous. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be selected from water, alcohol/aqueous solutions, emulsions, or suspensions (including saline and buffer media). Parenteral carriers include sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's glucose, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's glucose), etc. Preservatives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc., may also be present.

Antibody monomers, antibodies, antigen-binding fragments, and their immunoconjugates can be combined with therapeutic agents or compositions containing therapeutic agents for therapeutic purposes.

In some embodiments, the multispecific antibody molecule is used in combination with one or more additional therapeutic agents in an effective amount. The additional therapeutic agents include antibodies, chemotherapeutic agents, enzymes, or combinations thereof. In some embodiments, the additional therapeutic agent may be an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a DNA, RNA, or protein synthesis inhibitor, a RAS inhibitor, or a combination thereof. In some embodiments, the additional therapeutic agent may be a checkpoint inhibitor. In some embodiments, the therapeutic agent includes inhibitors of the following: PD1, PDL1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D, CD73, derivatives thereof, or combinations thereof.

In one embodiment, the therapeutic agent may include capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testosterone, voroxycycline, formexane, fazodazole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, NaB-paratacel, derivatives thereof, or combinations thereof. In one implementation, the therapeutic agents may include capecitabine, cisplatin, cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, cyclophosphamide, nitrogen mustard, vincristine, procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, epirubicin, pemetrexed, folic acid, gemcitabine, oxaliplatin, irinotecan, topotecan, camptothecin, docetaxel, paclitaxel, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testrolide, voroxycycline, and fazodone. Rozoflurane, Formexane, Fazodazole, Erlotinib, Lafatinib, Dasatinib, Gefitinib, Osimermetinib, Van der Tinib, Afatinib, Imatinib, Pazopinib, Lapatinib, Sunitinib, Nilotinib, Sorafenib, NaB-Palatase, Everolimus, Tessiromolimus, Dabufenib, Vermorafenib, Trametinib, Vinpocetine, Apatinib, Crizotinib, Periforsine, Olaparib, Bortezomib, Tofatinib, and their derivatives or combinations thereof.

Cancers, including breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer, gastric cancer, bladder cancer, cervical cancer, or brain cancer, can express cancer-related genes. Inhibiting cancer-related activity with specific monoclonal antibodies or antigen-binding fragments can have a therapeutic effect on cancer. Furthermore, administering therapeutically effective amounts of compositions containing monoclonal antibodies or antigen-binding fragments specific to cancer-related proteins can cure, prevent, improve, and delay the development or metastasis of cancer through cytotoxic action.

This disclosure can be more readily understood by referring to the following detailed description of specific embodiments and examples included herein. Although the invention has been described in detail with reference to certain embodiments thereof, such details should not be construed as limiting the scope of the invention.

Example

Example 1) Binding of a four-specific antibody to the EGFRvIII antigen

The binding of the four specific antibodies listed in Table 1 to the EGFRvIII antigen expressed on the surface of the U87 cell line was evaluated using FACS methods. The four specific antibodies were incubated with the U87 cell line and then detected using a second anti-human antibody directly conjugated with Alexa Fluor 647 fluorescent dye. Cell binding of the four specific antibodies was analyzed on a BD LSR Tortessa flow cytometer. All tested antibodies bound to the antigen in single-digit and sub-nanomolar KD ranges (Table 2). Observed differences in binding ranged from 3-fold and were likely driven by the position of the intramolecular binding domain and interactions with adjacent domains.

Table 1 shows examples of four-specific antibodies with an EGFRvIII tumor antigen-binding domain. Table 2 shows the binding to the EGFRvIII antigen expressed in the U87 cell line. The binding of the four-specific antibodies listed in Table 1 to the EGFRvIII antigen was evaluated by flow cytometry.

Table 1. Four specific antibodies with EGFRvIII tumor antigen-binding domain.

Table 2. Binding to EGFRvIII antigen expressed in CHO cell lines.

Example 2): Binding of four specific antibodies to EGFRvIII, 4-1BB, PD-L1 and CD3 protein antigens

The binding affinity and kinetics of the four specific antibodies listed in Table 1 to their respective antigens were evaluated using surface plasmon resonance on a Fortebio Octet RED96 instrument. The antigens were immobilized on the sensor chip surface, and the antibodies being tested flowed through the immobilized antigens. All molecules showed high binding to the antigens (Table 3). SI-39E29, SI-39E18, and SI-39E23 showed lower binding to the CD3 e/d antigen than the other antibodies tested. Table 3 shows the binding of the four specific antibodies listed in Table 1 to the EGFRvIII, 4-1BB, PD-L1, and CD3 antigens.

Table 3. Binding with EGFRvIII, 4-1BB, PD-L1 and CD3 antigens.

Table 3A.

Table 3B.

Example 3) Redirected PBMC cytotoxicity against EGFRvIII-transfected astrocytoma cell line U87

The ability of the four specific antibodies listed in Table 1 to redirect PBMC lysis in U87 cells transfected with the EGFRvIII tumor cell line (U87vIII) was evaluated. PBMCs were isolated by Ficoll gradient. The U87vIII tumor cell line stably expressed nuclear localization red fluorescent protein (RFP) delivered via lentiviral transduction (Sartorius). U87vIII tumor cells were co-cultured with PBMCs. Tumor cell lysis was evaluated by counting RFP-labeled tumor cell nuclei. Images were acquired on the IncuCyte (Sartorius) live-cell imaging system. The four specific antibodies SI-39E18 and SI-39E13 showed the highest potency at 96 hours, followed by SI-39E10. SI-39E4, SI-39E23, and SI-39E29 showed lower potency in this study than the other antibodies listed in Table 1 (Figure 2).

Example 4) Redirected PBMC cytotoxicity against the Kasumi-2 acute lymphoblastic leukemia cell line

The ability of the tetraspecific antibodies listed in Table 4 to lyse leukemia cells was evaluated. PBMCs were isolated using a Ficoll gradient. Kasumi-2 tumor cells were co-cultured with PBMCs. Tumor cell lysis was assessed using a BD LSR Tortesa flow cytometer by counting the number of viable tumor cells remaining after 96 hours of co-culture. The tetraspecific antibody SI-38E14 showed the most potent activity in this study, followed by SI-38E38 (Figure 3). Table 4 shows examples of tetraspecific antibodies with a CD19 tumor antigen recognition domain.

Table 4. Four specific antibodies with CD19 tumor antigen recognition domain.

Antibody ID Antibody domain structure SI-38E14 PL230×466F6×21D4×284A10 SI-38E38 PD224×466F6×21D4×284A10 SI-38E5 466F6×PL230×284A10×21D4 SI-38E20 466F6×21D4×284A10×PL221 SI-38E35 21D4×284A10×466F6×PL221

Example 5) Redirected PBMC cytotoxicity against EGFRvIII-transfected astrocytomas U87, with different 4-1BB... Functional activity of the domains and their functional impact on PD-L1 and 4-1BB domains

The ability of the four specific antibodies listed in Table 5 to redirect PBMC lysis in U87 cells transfected with the EGFRvIII tumor cell line (U87vIII) was evaluated. PBMCs were isolated by Ficoll gradient. The U87vIII tumor cell line stably expressed nuclear localization red fluorescent protein (RFP) delivered via lentiviral transduction (Sartorius). U87vIII tumor cells were co-cultured with PBMCs. Tumor cell lysis was assessed by counting RFP-labeled tumor cell nuclei. Images were obtained on the IncuCyte (Sartorius) live-cell imaging system. Antibody activity was evaluated after 96 hours of incubation. Antibodies with different 4-1BB domains—SI-39E4, SI-39E2, and SI-39E3—showed similar activity (Figure 4). Antibodies with PD-L1 and 4-1BB domains replaced by the silenced (non-functional) FITC domain, SI-39E1, and SI-39E5 showed reduced lysis activity. This observation confirms the functional contribution of the 4-1BB and PD-L1 domains. Table 5 shows examples of four-specific antibodies with the EGFRvIII tumor antigen-binding domain. FITC control antibody.

Table 5. Four-specific antibodies with EGFRvIII tumor antigen-binding domain. FITC control antibody.

Example 6) FACS analysis of four specific antibodies binding to human ROR1-transfected CHO cells

The binding of the four specific antibodies listed in Tables 1 and 2 to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1 was tested. A final concentration of 2X antibody was prepared and titrated 1:5 in three wells of a 96-well plate containing 50 μl PBS/2% FBS. Then, 5,000 ROR1-CHO cells were added to the plate in 50 μl PBS/2% FBS. The mixture was incubated on ice for 30 min, washed once with 200 μl PBS/2% FBS, and then a 1:1000 dilution of the secondary antibody PE goat anti-human IgG Fc was added. The mixture was incubated on ice for 30 min. The cells were washed with 2×200 μl PBS/2% FBS, resuspended in 50 μl PBS/2% FBS, and analyzed on a BD LSFORTESSA spectrophotometer. The binding profile is shown in Figure 5. The tetraspecific antibodies SI-35E18, 19, and 20, which have a 323H7 binding domain specific to the Ig domain of ROR1, showed higher binding than the tetraspecific antibodies SI-3521, 22, and 23, which have a 338H4 binding domain specific to the coiled domain of ROR1. Furthermore, the tetraspecific antibodies SI-3524, 25, and 26, which have a 330F11 binding domain specific to the kringle domain of ROR1, did not bind.

Example 7) FACS analysis of four specific antibodies binding to human 41BB-transfected CHO cells

The binding of the four specific antibodies listed in Table 6 to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1 was tested. A final concentration of 2X antibody was prepared and titrated 1:5 in three wells of a 96-well plate containing 50 μl PBS/2% FBS. Then, 5,000 ROR1-CHO cells were added to the plate in 50 μl PBS/2% FBS. The mixture was incubated on ice for 30 min, washed once with 200 μl PBS/2% FBS, and then a 1:1000 dilution of the secondary antibody PE goat anti-human IgG Fc was added. The mixture was incubated on ice for 30 min. The cells were washed with 2×200 μl PBS/2% FBS, resuspended in 50 μl PBS/2% FBS, and analyzed on a BD LSR Tortessa. The binding profile is shown in Figure 6. All four-specific antibodies, except for the control SI-27E12, contain the 41BB binding domains 460C3, 420H5, or 466F6 and bind to CHO cells expressing 41BB with varying strengths. Table 6 shows a list of exemplary four-specific antibodies.

Table 6. List of four specific antibodies.

Example 8) FACS analysis of four specific antibodies binding to human PD-L1 transfected CHO cells

The binding of the four specific antibodies listed in Table 6 to Chinese hamster ovary (CHO) cells stably expressing full-length human ROR1 was tested. A final concentration of 2X antibody was prepared and titrated 1:5 in three wells of a 96-well plate containing 50 μl PBS/2% FBS. Then, 5,000 ROR1-CHO cells were added to the plate in 50 μl PBS/2% FBS. The mixture was incubated on ice for 30 min, washed once with 200 μl PBS/2% FBS, and then a 1:1000 dilution of the secondary antibody PE goat anti-human IgG Fc was added. The mixture was incubated on ice for 30 min. The cells were washed with 2×200 μl PBS/2% FBS, resuspended in 50 μl PBS/2% FBS, and analyzed on a BD LSFORTESSA spectrophotometer. The binding profile is shown in Figure 7. All four specific antibodies, except for the control SI-27E15, contain the same PD-L1 binding domain PL230C6 and show binding strength very similar to that of CHO cells expressing PD-L1.

Example 9) Using peripheral blood mononuclear cells as the effector, B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2-targeted redirected T cell cytotoxicity (RTCC) assay

Human peripheral blood mononuclear cells (PBMCs) were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 mL of medium at 37 °C for 20 min. Cells were washed three times with 50 mL of medium, resuspended in 10 mL, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 10 wells of a 96-well plate in 200 μl RPMI + 10% FBS. Human PBMCs were purified from “leukopak,” an enriched leukocyte apheresis product collected from peripheral blood of normal individuals, using a standard Ficoll density gradient. In the final target 96-well plate, target cells, PBMCs, and serially titrated antibodies were combined by adding 100 μl of target cells (5,000), 50 μl of PBMCs (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, and then the contents of each well were collected and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 8, the four specific antibodies all contained the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 323H7, and the same CD3 binding domain 284A10, but had one of the 41BB binding domains 460C3, 420H5, and 466F6, and showed higher RTCC activity compared to controls other than SI-27E12, which did not have a 41BB binding domain but appeared to have similar potency to the four specific antibodies SI-35E18, 19, and 20.

Example 10) Using CD8+, CD45RO+ memory T cells as effectors, B-acute lymphoblastic leukemia (B-ALL) Redirected T cell cytotoxicity (RTCC) assay targeting Kasumi-2 cell line

Human CD8+, CD45RO+ memory T cells were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 ml of medium at 37°C for 20 min. Cells were washed three times with 50 ml of medium, resuspended in 10 ml, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 200 μl RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RO+ memory T cells were enriched from peripheral blood mononuclear cells from normal donors using the EasySep ^™ Human Memory CD8+ T Cell Enrichment Kit (StemCell Tech NO.logies, #19159) according to the manufacturer's protocol. FACS analysis confirmed a final cell population of 98% CD8+, CD45RO+ T cells (data not shown). In the final target 96-well plate, target cells, T cells, and serially titrated antibodies were pooled by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ memory T cells (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, after which the contents of each well were collected, and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 9, the four specific antibodies all contain the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 323H7, and the same CD3 binding domain 284A10, but have one of the 41BB binding domains 460C3, 420H5, and 466F6, and show higher RTCC activity compared to the control that does not contain one of the 41BB, PD-L1, ROR1, or CD3 binding domains.

Example 11) Using CD8+, CD45RA+ naïve T cells as effectors, B-acute lymphoblastic leukemia (B-ALL) Redirected T cell cytotoxicity (RTCC) assay targeting Kasumi-2 cell line

Human CD8+, CD45RA+ memory T cells were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 ml of medium at 37°C for 20 min. Cells were washed three times with 50 ml of medium, resuspended in 10 ml, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 200 μl RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RA+ memory T cells were enriched from peripheral blood mononuclear cells from normal donors using the EasySep ^™ Human Initial CD8+ T Cell Isolation Kit (StemCell Technology, #19258) according to the manufacturer's protocol. FACS analysis confirmed a final cell population of 98% CD8+, CD45RA+ T cells (data not shown). In the final target 96-well plate, target cells, T cells, and serially titrated antibodies were pooled by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RA+ T cells (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, after which the contents of each well were collected, and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 10, the four specific antibodies all contain the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 323H7, and the same CD3 binding domain 284A10, but have one of the 41BB binding domains 460C3, 420H5, and 466F6, and show higher RTCC activity compared to the control that does not contain one of the 41BB, PD-L1, ROR1, or CD3 binding domains.

Example 12) Using peripheral blood mononuclear cells as the effector and B-acute lymphoblastic leukemia (B-ALL) cell line Kasumi-2-targeted redirected T cell cytotoxicity (RTCC) assay

Human peripheral blood mononuclear cells (PBMCs) were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 mL of medium at 37 °C for 20 min. Cells were washed three times with 50 mL of medium, resuspended in 10 mL, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 10 wells of a 96-well plate in 200 μl RPMI + 10% FBS. Human PBMCs were purified from “leukopak,” an enriched leukocyte apheresis product collected from peripheral blood of normal individuals, using a standard Ficoll density gradient. In the final target 96-well plate, target cells, PBMCs, and serially titrated antibodies were combined by adding 100 μl of target cells (5,000), 50 μl of PBMCs (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, and then the contents of each well were collected and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 11, the four specific antibodies all contained the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 338H4, and the same CD3 binding domain 284A10, but had one of the 41BB binding domains 460C3, 420H5, and 466F6, and showed higher RTCC activity compared to controls other than SI-35E36, which did not have a 41BB binding domain but appeared to have similar potency to the four specific antibodies SI-35E18, 19, and 20.

Example 13) Using CD8+, CD45RO+ memory T cells as effectors, B-acute lymphoblastic leukemia (B-ALL) Redirected T cell cytotoxicity (RTCC) assay targeting Kasumi-2 cell line

Human CD8+, CD45RO+ memory T cells were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 ml of medium at 37°C for 20 min. Cells were washed three times with 50 ml of medium, resuspended in 10 ml, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 200 μl RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RO+ memory T cells were enriched from peripheral blood mononuclear cells from normal donors using the EasySep ^™ Human Memory CD8+ T Cell Enrichment Kit (StemCell Tech NO.logies, #19159) according to the manufacturer's protocol. FACS analysis confirmed a final cell population of 98% CD8+, CD45RO+ T cells (data not shown). In the final target 96-well plate, target cells, T cells, and serially titrated antibodies were pooled by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RO+ memory T cells (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, after which the contents of each well were collected, and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 12, the four specific antibodies all contain the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 338H4, and the same CD3 binding domain 284A10, but have one of the 41BB binding domains 460C3, 420H5, and 466F6, and show higher RTCC activity compared to the control that does not contain one of the 41BB, PD-L1, ROR1, or CD3 binding domains.

Example 14) Using CD8+, CD45RA+ naïve T cells as effectors, B-acute lymphoblastic leukemia (B-ALL) Redirected T cell cytotoxicity (RTCC) assay targeting Kasumi-2 cell line

Human CD8+, CD45RA+ memory T cells were used as effectors to test the RTCC activity of the four specific antibodies listed in Table 6 against the B-ALL cell line Kasumi2. Kasumi2 target cells, 5 × 10⁶, were labeled with CFSE (Invitrogen, #C34554) at 0.5 μM in 10 ml of medium at 37°C for 20 min. Cells were washed three times with 50 ml of medium, resuspended in 10 ml, and counted again. Antibodies were prepared to a final concentration of 2X and titrated 1:3 in 200 μl RPMI + 10% FBS in 10 wells of a 96-well plate. Human CD8+, CD45RA+ memory T cells were enriched from peripheral blood mononuclear cells from normal donors using the EasySep ^™ Human Initial CD8+ T Cell Isolation Kit (StemCell Technology, #19258) according to the manufacturer's protocol. FACS analysis confirmed a final cell population of 98% CD8+, CD45RA+ T cells (data not shown). In the final target 96-well plate, target cells, T cells, and serially titrated antibodies were pooled by adding 100 μl of target cells (5,000), 50 μl of CD8+, CD45RA+ T cells (25,000), and 100 μl of each antibody dilution to each well. The plate was incubated at 37°C for approximately 72 hours, after which the contents of each well were collected, and the number of residual CFSE-labeled target cells was analyzed. As shown in Figure 13, the four specific antibodies all contain the same PD-L1 binding domain PL230C6, the same ROR1 binding domain 338H4, and the same CD3 binding domain 284A10, but also possess one of the 41BB binding domains 460C3, 420H5, and 466F6. However, compared to the control that does not contain any of the 41BB, PD-L1, ROR1, or CD3 binding domains, they did not show greater RTCC activity. This contrasts with the four specific antibodies described in Example 6 and shown in Figure 10, which indeed showed RTCC activity against CD8+, CD45RA+ naive T cells.

While the invention has been described with reference to specific embodiments or examples, it should be understood that these embodiments are illustrative and the scope of the invention is not limited thereto. Alternative embodiments of this disclosure will be apparent to those skilled in the art to which this disclosure pertains. These alternative embodiments are considered to be included within the scope of this disclosure. Therefore, the scope of the invention is defined by the appended claims and supported by the foregoing description.

All references cited or mentioned in this disclosure are incorporated herein by reference in their entirety.

sequence list

CDRs in the amino acid sequence are underlined.

>SEQ ID:01 Anti-CD3 284A10 VHv1 nt

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCG AGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACGGTGGATCATCTGCTATTACTAGTAACAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCA

>SEQ ID:02 Anti-CD3 284A10 VHv1 aa

EVQLVESGGGLVQPGGSLRLSCAASGFTIS TNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAITSNN IWGQGTLVTVSS

>SEQ ID:03 Anti-CD3 284A10 VLv1 nt

GACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCT GGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:04 Anti-CD3 284A10 VLv1 aa

DVVMTQSPSTLSSASVGDRVTINC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNS FGGGTKVEIK

>SEQ ID:05 Anti-CD3 480C8 VHv1 nt

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAATCGACCTCAGTAGCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCG AGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACGGTGGATCATCTGCTATTAATAGTAAGAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCA

>SEQ ID:06 Anti-CD3 480C8 VHv1 aa

EVQLVESGGGLVQPGGSLRLSCAASGIDLS SNAMS WVRQAPGKGLEWIG VITGRDITYYASWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DGGSSAINSKNI WGQGTLVTVSS

>SEQ ID:07 Anti-CD3 480C8 VLv1 nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCT GGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:08 Anti-CD3 480C8 VLv1 aa

DIQMTQSPSTLSASSVGDRVTITC QASESISSWLA WYQQKPGKAPKLLIY EASKLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGYFYFISRTYVNA FGGGTKVEIK

>SEQ ID:09 Anti-PD-L1 PL230C6 VHv3 nt

CAGTCGGTGGAGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTACTTATAGTGGTAGTAGATACT ACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCTAGT

>SEQ ID:10 Anti-PD-L1 PL230C6 VHv3 aa

QSVEESGGGLVQPGGSLRLSCTASGIDL NTYDMI WVRQAPGKGLEWVG IITYSGSRYYANWAKG RFTISKDNTKNTVYLQMNSLRAEDTAVYYCAR DYMSGSHL WGQGTLVTVSS

>SEQ ID:11 Anti-PD-L1 PL230C6 VLv2 nt

GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTGGCA TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:12 Anti-PD-L1 PL230C6 VLv2 aa

AYDMTQSPSSVSASVGDRVTIKCQASEDI YSFLAWY QQKPGKAPKLLIH SASSLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQGYGKNNVDNA FGGGTKVEIK

>SEQ ID:13 Anti-PD-L1 PL221G5 VHv1 nt

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCGGGTACGACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTGCTGCTGGTAGTGCTGGTATCACTTAC GACGCGAACTGGGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGATCGGCGTTTTCGTTCGACTACGCCATGGACCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:14 anti-PD-L1 PL221G5 VHv1 aa

EVQLLESGGGLVQPGGSLRLSCAASGFSFS SGYDMC WVRQAPGKGLEWIA CIAAGSAGITYDANWAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SAFSFDYAMDL WGQGTLVTVSS

>SEQ ID:15 Anti-PD-L1 PL221G5 VLv1 nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTAGTTCCCACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCACTCTGGCA TCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTTACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGGGTTATAGTTGGGGTAATGTTGATAATGTTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:16 Anti-PD-L1 PL221G5 VLv1 aa

DIQMTQSPSTLSASSVGDRVTITC QASQSISSHLN WYQQKPGKAPKLLIY KASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQGYSWGNVDNV FGGGTKVEIK

>SEQ ID:17 Anti-PD-1PD224D1 VHv2 nt

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCTCCCTAAGTAGCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGCTACATTGGTGATACTACTGGCATAG CCTACGCGAGCTGGGCGAATGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGCTGGTCCTACTTAGACATCTGGGGCCAAGGGACCTGGTCACCGTCTCGAGC

>SEQ ID:18 Anti-PD-1PD224D1 VHv2 aa

EVQLVESGGGLVQPGGSLRLSCTASGFSLS SYAMS WVRQAPGKGLEYIG YIGDTTGIAYASWANG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR GWSYLDI WGQGTLVTVSS

>SEQ ID:19 Anti-PD-1PD224D1 VLv2 nt

GCCCTTGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAACATTTACAGCAATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATCAGGCCTCCACTCTGG CATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATATGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAGGCGGTTATTATAGTGCTGCCCTTAATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:20 Anti-PD-1PD224D1 VLv2 aa

ALVMTQSPSSSLSASVGDRVTITC QASQNIYSNLA WYQQKPGKVPKLLIY QASTLAS GVPSRFSGSGYGTDFTLTISSLQPEDVATYYC QGGYYSAALNT FGGGTKVEIK

>SEQ ID:21 Anti-4-1BB 420H5 VHv3 nt

CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCTTCAGTAGCAACTACTGGATATGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATTTATGTTGGTAGTAGTGGTGACACTTAC TACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATAGTAGTAGTTATTATATGTTTAACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:22 Anti-4-1BB 420H5 VHv3 aa

QSLVESGGGLVQPGGSLRLSCAASGFSFS SNYWIC WVRQAPGKGLEWIA CIYVGSSGDTYYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DSSSYYMFNL WGQGTLVTVSS

>SEQ ID:23 Anti-4-1BB 420H5 VLv3 nt

GCCCTTGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTGAGGACATTGATAACCTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTTTATGCATCCGATCTGGCATCT GGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCGGTTACTATACTAGTAGTGCTGATACGAGGGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:24 Anti-4-1BB 420H5 VLv3 aa

ALVMTQSPSTLSASSVGDRVTINC QASEDIDTYLA WYQQKPGKAPKLLIF YASDLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QGGYYTSSADTRGA FGGGTKVEIK

>SEQ ID:25 anti-4-1BB 466F6 VHv2 nt

CGGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGATTCACCATCAGTAGCTACCACATGCAGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTACATCGGAACCATTAGTAGTGGTGGTAATGTATACT ACGCGAGCTCCGCGAGAGGCAGATTCACCATCTCCAGACCCTCGTCCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACTCTGGTTATAGTGATCCTATGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:26 Anti-4-1BB 466F6 VHv2 aa

RSLVESGGGLVQPGGSLRLSCTASGFTIS SYHMQ WVRQAPGKGLEYIG TISSGGNVYYASSARG RFTISRPSSKNTVDLQMNSLRAEDTAVYYCAR DSGYSDPM WGQGTLVTVSS

>SEQ ID:27 Anti-4-1BB 466F6 VLv5 nt

GACGTTGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACCTGTCAGGCCAGTCAGAACATTAGGACTTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAGCCAATCTGGCAT CTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGACCTGGAGCCTGGCGATGCTGCAACTTACTATTGTCAGTCTACCTATCTTGGTACTGATTATGTTGGCGGTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:28 Anti-4-1BB 466F6 VLv5 aa

DVVMTQSPSSVSASVGDRVTITC QASQNIRTYLS WYQQKPGKAPKLLIY AAANLAS GVPSRFSGSGSGTDFTLTISDLEPGDAATYYC QSTYLGTDYVGGA FGGGTKVEIK

>SEQ ID:29 Anti-4-1BB 460C3 VHv1 nt

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAATCGACTTCAGTAGGAGATACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATATATACTGGTAGCCGCG ATACTCCTCACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGAAGGTAGCCTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGC

>SEQ ID:30 anti-4-1BB 460C3 VHv1 aa

EVQLLESGGGLVQPGGSLRLSCAASGIDFS RRYYMC WVRQAPGKGLEWIA CIYTGSRDTPHYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR EGSL WGQGTLVTVSS

>SEQ ID:31 Anti-4-1BB 460C3 VLv1 nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAGTAACTGGTTCTCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTCTGG CATCTGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCGCAGGCGGTTACAATACTGTTATTGATACTTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:32 Anti-4-1BB 460C3 VLv1 aa

DIQMTQSPSTLSASVGDRVTITC QSSQSVYSNWFS WYQQKPGKAPKLLIY SASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC AGGYNTVIDTFA FGGGTKVEIK

>SEQ ID:33 Anti-ROR1 324C6 VHv2 nt

CAGTCGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCTCCCTCAGTAGGTACTACATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTTATACTAGTGGTAGTACATGGTACGCGA GCTGGACAAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATCCTATTATGGCGGTGATAAGACTGGTTTAGGCATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA

>SEQ ID:34 Anti-ROR1 324C6 VHv2 nt

QSLVESGGGLVQPGGSLRLSCTASGFSLS RYYMT WVRQAPGKGLEWIG TIYTSGSTWYASWTKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR SYYGGDKTGLGI WGQGTLVTVSS

>SEQ ID:35 anti-ROR1 324C6 VLv1 nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCCAGTCAGAGCATTGATAGTTGGTTATCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAGGCATCCACTCTGGCATCTG GGGTCCCATCAAGGTTCAGCGGCAGTGGATTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAATCTGCTTATGGTGTTAGTGGTACTAGTAGTTATTTATATACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:36 Anti-ROR1 324C6 VLv1 aa

DIQMTQSPSTLSASVGDRVTITC QASQSIDSWLS WYQQKPGKAPKLLIY QASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSAYGVSGTSSYLYT FGGGTKVEIK

>SEQ ID:37 anti-ROR1 323H7 VHv4 nt

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGACATATTTATGTTAATAATGATGACACAGACTACGCG AGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCACCTATTTCTGTGCGAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA

>SEQ ID:38 anti-ROR1 323H7 VHv4 aa

EVQLLESGGGLVQPGGSLRLSCAAS GFTISRYHMT WVRQAPGKGLEWIG HIYVNNDDTDYASSAKG RFTISRDNSKNTLYLQMNSLRAEDTATYFCAR LDVGGGGAYIGDI WGQGTLVTVSS

>SEQ ID:39 anti-ROR1 323H7 VLv1 nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCTTCCACTCTGG CATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:40 anti-ROR1 323H7 VLv1 aa

DIQMTQSPSSSLSASVGDRVTITC QSSQSVYNNNDLA WYQQKPGKVPKLLIY YASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC AGGYDTDGLDTFA FGGGTKVEIK

>SEQ ID:41 Anti-ROR1 338H4 VHv3 nt

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACTGCCTCTGGATTCTCCCTCAGTAGCTATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAGGGGGCTGGAGTGGATCGGAATCATTTATGCTAGTGGTAGCACAT ACTACGCGAGCTCGGCGAAAGGCAGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGGATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAATTTATGACGGCATGGACCTCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA

>SEQ ID:42 Anti-ROR1 338H4 VHv3 aa

EVQLVESGGGLVQPGGSLRLSCTASGFSL SSYAMS WVRQAPGRGLEWIG IIYASGSTYYASSAKG RFTISKDNTKNTVDLQMNSLRAEDTAVYYCAR IYDGMDL WGQGTLVTVSS

>SEQ ID:43 anti-ROR1 338H4 VLv4 nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAGGCCAGTCAGAACATTTACAGCTACTTATCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCGCCTGATCTATCTGGCATCTACTC TGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTACACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGCAATTATAACGGTAATTATGGTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:44 Anti-ROR1 338H4 VLv4 aa

DIQMTQSPSSSLSASVGDRVTINC QASQNIYSYLS WYQQKPGKVPKRLIY LASTLAS GVPSRFSGSGSGTDYTLTISSLQPEDVATYYC QSNYNGNYG FGGGTKVEIK

>SEQ ID:45 anti-ROR1 330F11 VHv1 nt

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCCCTCAATAACTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAACCATTAGTAGTGGTGCGTATACATGGTTCGCC ACCTGGGCGACAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGATATTCTTCTACTACTGATTGGACCTACTTTAACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCA

>SEQ ID:46 Anti-ROR1 330F11 VHv1 aa

EVQLVESGGGLVQPGGSLRLSCAASGFSLN NYWMS WVRQAPGKGLEWIG TISSGAYTWFATWATG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR YSSTTDWTYFNI WGQGTLVTVSS

>SEQ ID:47 Anti-ROR1 330F11 VLv1 nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGGGCATCCACTCTG GAATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAAGCTATAATGGTGTTGGTAGGACTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:48 Anti-ROR1 330F11 VLv1 aa

DIQMTQSPSTLSASVGDRVTITC QASQSINNYLA WYQQKPGKAPKLLIY RASTLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSYNGVGRTA FGGGTKVEIK

>SEQ ID:49 anti-EGFRvIII mAb 806VH nt

GATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCTTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAACTGGATTCGGCAGTTTCCAGGAAACAAGCTGGAGTGGATGGGCTACATAAGTTATAGTGGTAACACT AGGTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGCGACACATCCAAGAACCAATTCTTCCTGCAGTTGAACTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCGGGACGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

>SEQ ID:50 Anti-EGFRvIII mAb 806VH aa

DVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG YISYSGNTRYNPSLKS RISITRDTSKNQFFLQLNSVTIEDTATYYCVT AGRGFPY WGQGTLVTVSA

>SEQ ID:51 anti-EGFRvIII mAb 806VL nt

GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACT TGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

>SEQ ID:52 Anti-EGFRvIII mAb 806VL aa

DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIK

>SEQ ID:53 anti-CD19 21D4 VH nt

GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAACCAGGAGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTAGCAGTTCATGGATCGGCTGGGTGCGCCAGGCACCTGGGAAAGGCCTGGAATGGATGGGGATCATCTATCCTGATGACTCTGATACCAGATACA GTCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGGACTGCCTACCTGCAGTGGAGTAGCCTGAAGGCCTCGGACACCGCTATGTATTACTGTGCGAGACATGTTACTATGATTTGGGGAGTTATTATTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

>SEQ ID:54 anti-CD19 21D4 VH aa

EVQLVQSGAEVKKPGESLKISCKGSGYSFS SSWIG WVRQAPGKGLEWMG IIYPDDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASDTAMYYCAR HVTMIWGVIIDF WGQGTLVTVSS

>SEQ ID:55 anti-CD19 21D4 VL nt

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTT TGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

>SEQ ID:56 anti-CD19 21D4 VL aa

AIQLTQSPSSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQFNSYPFT FGPGTKVDIK

>SEQ ID:57 Anti-FITC 4-4-20VH nt

GAGGTGAAGCTGGATGAGACTGGAGGAGGCTTGGTGCAACCTGGGAGGCCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAAGGACTGGAGTGGGTAGCACAAATTAGAAACAAACCTTATAATTATGAA ACATATTATTCAGATTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGTTGAAGACATGGGTATCTATTACTGTACGGGTTCTTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

>SEQ ID:58 anti-FITC 4-4-20VH aa

EVKLDETGGGLVQPGRPMKLSCVASGFTFS DYWMN WVRQSPEKGLEWVA QIRNKPYNYETYYSDSVKG RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTG SYYGMDY WGQGTSVTVSS

>SEQ ID:59 anti-FITC 4-4-20VL nt

GATGTCGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACGTTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGGTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

>SEQ ID:60 anti-FITC 4-4-20VL aa

DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLR WYLQKPGQSPKVLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK

>SEQ ID: 61 human IgG1 deletion (null) (G1m-fant with ADCC/CDC deletion mutation (null mutation))

GCTAGCACCAAGGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT

>SEQ ID: 62 Human IgG1 invalid (G1m-fa with ADCC/CDC invalid mutation)aa

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

>SEQ ID: 63 people Igκnt

CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

>SEQ ID: 64 people Igκaa

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

>SEQ ID:65SI-35E18(60C3-L1H1-scFv×PL230C6-Fab×323H7-H4L1-scFv×284A10-H1L1-scFv) heavy chain nt

GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAGTAACTGGTTCTCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAG CAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCGCAGGCGGTTACAATACTGTTATTGATACTTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCAGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG CAGCCTCTGGAATCGACTTCAGTAGGAGATACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGCATATATACTGGTAGCCGCGATACTCCTCACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCG AGAGAAGGTAGCCTGTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGCGGTGGAGGGTCCGGCGGTGGTGGATCCCAGTCGGTGGAGGAGTCTGGGGGAGCTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTACAGCCTCTGGAATCGACCTTAATACCTACGACATGATCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAGTGGGTTGGAATCATTAC TTATAGTGGTAGTAGATACTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAGACAATACCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCCAGAGATTATATGAGTGGTTCCCACTTGTGGGGCCAGGGAACCCTGGTCACCGTCTCTAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCC AGCAACACCAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCGCGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTATACCCTGCCCCCATCCCGGGATGAGCTGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGCGGTGGAGGGTCCGGCGGTGGTGGATCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTCGCTACCACATGACTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGACATATTTATGTTAATAATGATGA CACAGACTACGCGAGCTCCGCGAAAGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCACCTATTTCTGTGCGAGATTGGATGTTGGTGGTGGTGGTGCTTATATTGGGGACATCTGGGGCCAGGGAACTCTGGTTACCGTCTCTTCAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCG GCGGAGGGTCCGGCGGTGGAGGATCAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGTCCAGTCAGAGTGTTTATAACAACAACGACTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTATTATGCTTCCACTCTGGCATCTGGGGTCCCATCTCGGTTCAGTGGCAGTGGA TCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTGCAGGCGGTTATGATACGGATGGTCTTGATACGTTTGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAAGGCGGTGGAGGGTCCGGCGGTGGTGGATCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTG TGCAGCCTCTGGATTCACCATCAGTACCAATGCAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCATTACTGGTCGTGATATCACATACTACGCGAGCTGGGCGAAAGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGCGACG GTGGATCATCTGCTATTACTAGTAACAACATTTGGGGCCAAGGAACTCTGGTCACCGTTTCTTCAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCAGACGTCGTGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAATTGCCAAGCCAGTGAGAGCATTAGCAGTTGGTTAGCCTGG TATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAAGCATCCAAACTGGCATCTGGGGTCCCATCAAGGTTCAGCGGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAAGGCTATTTTTATTTTATTAGTCGTACTTATGTAAATTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

>SEQ ID:66SI-35E18(460C3-L1H1-scFv×PL230C6-Fab×323H7-H4L1-scFv×284A10-H1L1-scFv) heavy chain aa

DIQMTQSPSTLSASVGDRVTITCQSSQSVYSNWFSWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGYNTVIDTFAFGGGTKVEIK(Anti-4-1BB 460C3 VLv1)

GGGGSGGGGSGGGGSGGGGS(Gly ₄ Ser)x4 connector

EVQLLESGGGLVQPGGSLRLSCAASGIDFSRRYYMCWVRQAPGKGLEWIACIYTGSRDTPHYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGSLWGQGTLVTVSS (Anti-4-1BB 460C3 VHv1)

GGGGSGGGGS(Gly ₄ Ser)x2 connector

QSVEESGGGLVQPGGSLRLSCTASGIDLNTYDMIWVRQAPGKGLEWVGIITYSGSRYYANWAKGRFTISKDNTKNTVYLQMNSLRAEDTAVYYCARDYMSGSHLWGQGTLVTVSS(anti-PD-L1 PL230C6 VHv3)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

HYTQKSLSLSPG (Human IgG1 ineffective)

GGGGSGGGGS(Gly ₄ Ser)x2 connector

EVQLLESGGGLVQPGGSLRLSCAASGFTISRYHMTWVRQAPGKGLEWIGHIYVNNDDTDYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTATYFCARLDVGGGGAYIGDIWGQGTLVTVSS(anti-ROR1 323H7 VHv4)

GGGGSGGGGSGGGGSGGGGS(Gly ₄ Ser)x4 connector

DIQMTQSPSSLSSASVGDRVTITCQSSQSVYNNNDLAWYQQKPGKVPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCAGGYDTDGLDTFAFGGGTKVEIK(anti-ROR1 323H7 VLv1)

GGGGSGGGGS(Gly ₄ Ser)x2 connector

EVQLVESGGGLVQPGGSLRLSCAASGFTISTNAMSWVRQAPGKGLEWIGVITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSS (Anti-CD3 284A10 VHv1)

GGGGSGGGGSGGGGSGGGGS(Gly ₄ Ser)x4 connector

DVVMTQSPSTLSASSVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGTKVEIK(Anti-CD3 284A10 VLv1)

>SEQ ID:67SI-35E18(460C3-L1H1-scFv×PL230C6-Fab×323H7-H4L1-scFv×284A10-H1L1-scFv) Light chain nt

GCCTATGATATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCAAGTGTCAGGCCAGTGAGGACATTTATAGCTTCTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATTCTGCATCCTCTCTG GCATCTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTTATGGTAAAAATAATGTTGATAATGCTTTCGGCGGAGGGACCAAGGTGGAGA TCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

>SEQ ID:68SI-35E18(460C3-L1H1-scFv×PL230C6-Fab×323H7-H4L1-scFv×284A10-H1L1-scFv) Light chain aa

AYDMTQSPSSVSASVGDRVTIKCQASEDIYSFLAWYQQKPGKAPKLLIHSASSLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYGKNNVDNAFGGGTKVEIK(anti-PD-L1 PL230C6 VLv2)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (human Igκ)

Other sequences:

Note: The EGFRvIII systemic immune sequence is missing the C-terminal arginine.

Sequence index:

Claims (20)

1. A tetraspecific antibody monomer having an N-terminus and a C-terminus, comprising, from the N-terminus to the C-terminus, the following components in sequence: The first scFv structural domain at the N-terminus, Fab domain, Fc structural domain The second scFv structural domain, and The third scFv structural domain at the C-terminus, The first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each have binding specificity for different antigens, and the antigen is a tumor antigen, an immune signaling antigen, or a combination thereof. 2. The four-specific antibody monomer according to claim 1, wherein the first scFv domain, the Fab domain and the second scFv domain each independently have binding specificity against an antigen, wherein the antigen is selected from ROR1, PD-L1, CD3, CD28, 41BB, CEA, HER2, EGFRvIII, EGFR, LMP1, LMP2A, mesothelin, PSMA, EpCAM, glypimay-3, gpA33, GD2, TROP2, NKG2D, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73 and CD39, and wherein the Fc domain comprises a human IgG Fc domain. 3. The tetraspecific antibody monomer according to claim 1, wherein from the N-terminus to the C-terminus it comprises, in sequence: The first scFv structural domain at the N-terminus, Fab domain, Fc structural domain The second scFv structural domain, and The third scFv structural domain at the C-terminus, The first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each have binding specificity for different antigens, and the antigen is a tumor antigen, an immune signaling antigen, or a combination thereof. The first scFv domain, the Fab domain, the second scFv domain, and the third scFv domain each independently have binding specificity for antigens selected from CD3, EGFRvIII, PD-L1, and 4-1BB. 4. The four-specific antibody monomer according to claim 1, wherein the first scFv domain has binding specificity against CD3. 5. The four-specific antibody monomer according to claim 1, wherein the Fab domain has binding specificity against EGRF VIII. 6. The four-specific antibody monomer according to claim 1, wherein the second scFv domain has binding specificity against PD-L1. 7. The four-specific antibody monomer according to claim 1, wherein the third scFv domain has binding specificity for 4-1BB. 8. The four-specific antibody monomer according to claim 1, wherein the first scFv domain has binding specificity against CD3, wherein the Fab domain has binding specificity against EGRF VIII, wherein the second scFv domain has binding specificity against PD-L1, and wherein the third scFv domain has binding specificity against 4-1BB. 9. The four-specific antibody monomer according to claim 1, wherein the first scFv domain, the second scFv domain or the third scFv domain comprises a gly-gly-gly-gly-ser(G4S) _n linker, wherein n is 2, 3 or 4. 10. The four-specific antibody monomer according to claim 1, comprising a heavy chain CDR sequence and a light chain CDR sequence, wherein the heavy chain CDR sequence is: TNAMS,VITGRDITYYASWAKG,DGGSSAITSNN,QASESISSWLA,EASKLAS,QGYFYFISRTYVNA,SDFAWN,YISYSGNTRYNPSLKS,AGRGFPY,SGYDMC,CIAAGSAGITYDANWAKG,SAFSFDYAMDL,QASQSISSHLN,KASTLAS,QQGYSWGNVDNV,SNYWIC,CIYVGSSGDTYYASSAKG,DSSSYYMFNL,QASEDIDTYLA,YASDLAS,QGGYYTSSADTRGA; and the light chain CDR sequence is: HSSQDINSNIG,HGTNLDD,VQYAQFPWT. 11. The tetraspecific antibody monomer according to claim 1, comprising the heavy chain variable region (HL) amino acid sequence and the light chain variable region (VL) amino acid sequence shown in SEQ ID NO. 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 58, 60, 62 and 64. 12. The tetraspecific antibody monomer according to claim 1, comprising a heavy chain variable region (HL) amino acid sequence and a light chain variable region (VL) amino acid sequence having percentage homology with the following amino acid sequences respectively: SEQ ID NO. 02, 04, 06, 08, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 50, 52, 54, 56, 58, 60, 62 and 64, wherein the percentage homology is not less than 98%. 13. A tetraspecific antibody comprising the tetraspecific antibody monomer according to claim 1. 14. An isolated nucleic acid encoding a tetraspecific antibody monomer according to any one of claims 1-12 or a tetraspecific antibody according to claim 13. 15. The nucleic acid of claim 14, comprising a nucleotide sequence having a percentage homology with any of the following nucleotide sequences: SEQ ID NO. 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 49, 51, 53, 55, 57, 59, 65 and 67, wherein the percentage of homology is not less than 98%. 16. An expression vector comprising the isolated nucleic acid sequence according to claim 14 or 15. 17. A host cell comprising the isolated nucleic acid according to claim 14 or 15, wherein the host cell is a prokaryotic cell or a eukaryotic cell. 18. A method for producing a tetraspecific antibody or monomer, comprising culturing a host cell of claim 17 to express a DNA sequence encoding the tetraspecific antibody or monomer, and purifying the tetraspecific antibody. 19. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tetraspecific antibody according to claim 13. 20. Use of the tetraspecific antibody of claim 13 in the preparation of a medicament for treating a human subject suffering from cancer.