Patent Application
20230113563
The present application generally relates to the technical field of multi-specific antibody for cancer immunotherapy and more particularly relates to making and using Guidance and Navigation Control (GNC) antibodies with multiple binding activities against surface molecules of both immune cells and tumor cells.
Cancer cells develop various strategies to evade the immune system. There is an urgent need to improve biological therapeutics functionality, specificity, potency, and efficacy. The success of targeted therapies in cancer treatment has also been impeded by various mechanisms of resistance. Tumor plasticity has emerged as a mode of targeted therapy evasion in various cancers, ranging from prostate and lung adenocarcinoma to melanoma and basal cell carcinoma. It is thought that the mechanisms interfering in shaping robust antitumor immune responses include at least some of the following categories: 1) defective tumor antigen processing or presentation; 2) lack of activating mechanisms; 3) inhibitory mechanisms and immunosuppressive state; and 4) resistant tumor cells (4). To overcome these mechanisms of escaping and resisting, new therapeutic strategies are designed to facilitate multiple immune effectors, including but not limited to, T-cell engager, checkpoint inhibitors, and innate immunity into the combination immunotherapy strategy. However, such strategies of combination therapy often mean two or more independent biologics products, which require manufacture of independent biologics as well as approval of the clinical safety and efficacy of each product. Combination therapies may target either immune cells or tumor cells or both. For instance, there are antibody therapies using bispecific antibodies targeting both CD3 and CD19 or CAR-T cell therapy comprising engineered T cells expressing anti-CD19 chimeric antibody. One common side effect from these immunotherapies is cytokine release syndrome, which is indicative of insufficient immune regulation. In this context, new strategies are needed to overcome tumor plasticity, i.e. the heterogeneous and dynamic expression of tumor antigens and/or resistant tumor cells, while acquiring additional immune regulation.
Towards this end, a platform of multi-specific antibodies, also known as Guidance and Navigation Control (GNC), has been established to facilitate multiple targeting of T-cell engagers, co-stimulation factors, checkpoint inhibitors, and tumor antigens (see Applicant's applications, WO/2019/005641, WO2019191120, and PCT/US20/59230, incorporated herein in its entirety). In addition, tetra-specific GNC (tetraGNC) antibodies may be used to manufacture GNC-T cell therapy for treating both liquid and solid tumors. In spite of multifunctional GNC molecules, epitope-negative tumor cells can remain un-targeted and consequently evade immunotherapies. For instance, the expression of NKG2D ligands are tightly regulated to prevent autoimmune tissue damage and therefore normal tissues generally do not express NKG2D ligands. Using the NKG2D receptor may therefore be an effective targeting mechanism for cancer immunotherapy via an innate immune recognition process. In this context, there is a clear need to further develop multi-specific antibodies related cell therapy. While a multi-specificity single drug remains highly desirable and cost-effective, it is technically challenging to design, express, and manufacture a potent and stable multi-specific antibody beyond tetra-GNC antibodies.
The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
The application provides proteins with binding specificities such as multi-specific antibody-like protein may include multi-specific antibodies, the fragments of these binding proteins may include without limitation scFv domain, Fab region, Fc domain, VH, VL, light chains, heavy chains, variable regions, and complementary determining region (CDR), methods of making and method using the multi-specific antibody-like proteins and fragments thereof.
In one embodiment, the multi-specific antibody-like protein may be multi-specific antibodies, monoclonal antibodies, isolated monoclonal antibodies, or humanized antibodies.
In one embodiment, the proteins may comprise various domains and regions such as binding domains. In one embodiment, the multi-specific antibody-like protein may include one or more binding domains including a first binding domain (D1), a second binding domain (D2), a third binding domain (D3), a fourth binding domain (D4), a fifth binding domain (D5), or a sixth binding domain (D6). The multi-specific antibody-like protein disclosed herein may be mono-specific, bi-specific, tri-specific, tetra-specific, penta-specific or hexa-specific.
In one embodiment, the binding domains such as D1, D2, D3, D4, D5, and D6 may each independently have a binding affinity to specificity against a T cell activating receptor, an immune cell receptor, an immune checkpoint molecule, a co-stimulation factor, a receptor of a leukocyte, a tumor antigen, a tumor associated antigen (TAA), a receptor of a tissue cell, a receptor of a cancer cell, or a combination thereof.
In one embodiment, the T cell activating receptor may comprise CD3. In one embodiment, the immune checkpoint receptor may comprise PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PD-L2, CD160, LOX-1, siglec-15, CD47, HVEM SIRPα, CSF1R, CD73, Siglec-15, CD47, or a combination thereof. In one embodiment, the co-stimulating receptor may comprise 4-1BB, CD28, OX40, GITR, CD40L, CD40, ICOS, LIGHT, CD27, CD30, or a combination thereof. In one embodiment, the tumor associated antigen may comprise EGFR, HER2, HER3, EGRFVIII, CD19, BCMA, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D ligand, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, uPAR, CD38, or a combination thereof.
In one embodiment, the binding domain for a T cell activating receptor is adjacent to the binding domain for a tumor associated antigen (TAA).
In one embodiment, the D1, D3, D4, D5, and D6 may be independently a scFv domain, a receptor, or a ligand. In one embodiment, at least one, two, three, four, or five of D1, D3, D4, D5, and D6 in the hexa-specific antibody-like protein include an scFv domain. In one embodiment, all of D1, D3, D4, D5, and D6 are all scFv domains.
In one embodiment, at least one, two, three, four, or five of D1, D3, D4, D5, and D6 in the hexa-specific antibody-like protein include a receptor. In one embodiment, all of D1, D3, D4, D5, and D6 are all receptors.
In one embodiment, at least one, two, three, four, or five of D1, D3, D4, D5, and D6 in the hexa-specific antibody-like protein include a ligand. In one embodiment, all of D1, D3, D4, D5, and D6 are all ligands.
In one embodiment, the scFv domain may comprise a VH linked to a VL in the orientation of VH-VL or VL-VH. In one embodiment, the scFv domain may comprise a disulphide bond between the VL and the VH. In one embodiment, the disulfide bond is between VL100 and VH44 of the scFv domain. In one embodiment, the scFv domain may comprise a substitution R19S (Kabat) in the VH.
In one embodiment, the multi-specific antibody-like protein may include a Fc region. In one embodiment, the Fc region is engineered to eliminate effector cell functions including without limitation ADCC, ADCP, or CDC. In one embodiment, the Fc region comprise at least one mutation at L234A, L235A, G237A, or K322A (Eu numbering). In one embodiment, the Fc region comprise mutations at L234A/L235A/G237A/K322A. In one embodiment, the Fc region comprise mutations at L234A/L235A/K322A (Eu numbering).
The domains and regions may be linked through linkers. In one embodiment, the linker may comprise a (GxSy)n linker, wherein n, x and y each independently is an integer from 1 to 10. In one embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In one aspect, the application provides hex-specific antibody-like proteins. In one embodiment, the hexa-specific antibody-like protein having a N-terminal and a C-terminal, may include in tandem from the N-terminal to the C-terminal, a first binding domain (D1) at the N-terminal, a Fab region as a second binding domain (D2) may include a light chain, a Fc region, a third binding domain (D3) having a binding affinity to PD-L1, and a fourth binding domain (D4) having a binding affinity to 4-1BB at the C-terminal, wherein the light chain may comprise a fifth binding domain (D5) covalently attached to the C-terminal and a sixth binding domain (D6) covalently attached to the N-terminal, and wherein D1, D2, D5, and D6 each independently may have a binding affinity to a tumor associated antigen (TAA) or CD3.
In one embodiment, the hexa-specific antibody-like protein may have D1 or D2 having binding affinity to CD3. In one embodiment, the hexa-specific antibody-like protein may have D1 having a binding affinity to CD3. In one embodiment, the hexa-specific antibody-like protein may have D2 having a binding affinity to CD3.
In one embodiment, the hexa-specific antibody-like protein may include D1 having a binding specificity against CD3, D2 having a binding specificity against EGFR, EGFRvIII, CD20, mesothelin, Claudin18.2, HER2, CD33or a combination thereof, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 and D6 each independently having a binding specificity against a tumor associated antigen.
In one embodiment, the hexa-specific antibody-like protein may include D1 having a binding specificity against CD3, D2 having a binding specificity against a tumor associated antigen, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 and D6 each independently having a binding specificity against NKG2D ligands, HER3, CD19 or a combination thereof.
In one embodiment, the hexa-specific antibody-like protein may include D1 having a binding specificity against EGFR, D2 having a binding specificity against CD3, D3 may have a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against CD19, and D6 having a binding specificity against HER3.
In one embodiment, the hexa-specific antibody-like protein having D1 may have a binding specificity against EGFR, D2 having a binding specificity against CD3, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against HER3, and D6 having a binding specificity against CD19.
In one embodiment, the hexa-specific antibody-like protein may include D1 having a binding specificity against CD3, D2 having a binding specificity against EGFR, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against HER3, and D6 having a binding specificity against CD19.
In one embodiment, the hexa-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of sequence identify to SEQ ID NO. 176, 178, 106, 108, 332, 334, 324, 326, 328, or 330.
In one aspect, the application provides tetra-specific or penta-specific antibody-like proteins. In one embodiment, the antibody-like protein having a N-terminal and a C-terminal, may include in tandem from the N-terminal to the C-terminal, a first binding domain (D1) at the N-terminal, a Fab region as a second binding domain (D2) may include a light chain, wherein the light chain optionally may comprise a fifth binding domain (D5) covalently attached to the C-terminal or a sixth binding domain (D6) covalently attached to the N-terminal, a Fc region, a third binding domain (D3), and a fourth binding domain (D4) at the C-terminal. In one embodiment, the multi-specific antibody-like protein comprise an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of sequence identify to SEQ ID NO. 110, 112, 116, 118, 122, 124, 128, 130, 134, 136, 140, 142, 146, 148, 152, 154, 158, 160, 164, 166, 170, 172, 112,114, 118, 120, 124, 126, 130, 132, 136, 138, 142, 144, 148, 150, 154, 156, 160, 162, 166, 168, 172, 174, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 302, 304, 306, or 308.
In one embodiment, the multi-specific antibody-like protein may tetra-specific. In one embodiment, the multi-specific antibody-like protein may penta-specific. In one embodiment, D2, D5, and D6 each independently may have a binding affinity to a tumor associate antigen (TAA).
In one embodiment, the tetra-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 having a binding specificity against a tumor-associated antigen, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the tetra-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 having a binding specificity against an antigen selected from a group consisting of EGFR, HER2, CD19, CD20, CD22, CD30, CD22, mesothelin, GD2, and Claudin 18.2, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the penta-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 and D5 independently each having a binding specificity against a tumor-associated antigen, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the penta-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 having a binding specificity against a tumor-associated antigen, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against HER3.
In one embodiment, the penta-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 having a binding specificity against EGFR or EGFRvIII, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against HER3.
In one embodiment, the penta-specific antibody-like protein may have D1 having a binding specificity against CD3, D2 having a binding specificity against CD20, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a binding specificity against CD19.
In one embodiment, the penta-specific antibody-like protein may have D1 and D6 independently having a binding specificity against a tumor-associated antigen, D2 having a binding specificity against CD3, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the penta-specific antibody-like protein may have D1 having a binding specificity against EGFR, D2 having a binding specificity against CD3, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D6 having a binding specificity against CD19.
In one aspect, the application provides multi-specific antibody-like protein with at least one binding domain as a receptor. In one embodiment, the receptor is NKG2D.
In one embodiment, the multi-specific antibody-like protein having a N-terminal and a C-terminal, may include in tandem from the N-terminal to the C-terminal, optionally a first binding domain (D1) at the N-terminal, a second binding domain (D2) may include a light chain, wherein the light chain optionally may comprise a fifth binding domain (D5) covalently attached to the C-terminal, a sixth binding domain (D6) covalently attached to the N-terminal, or both, a Fc region, optionally a third binding domain (D3), and optionally a fourth binding domain (D4) at the C-terminal, wherein at least one of D1, D2, D3, D4, D5, and D6 is a NKG2D, and wherein D1, D2, D3, D4, D5, and D6 each independently may have a binding affinity to specificity against a T cell activating receptor, an immune cell receptor, an immune checkpoint molecule, a co-stimulation factor, a receptor of a leukocyte, a tumor antigen, a tumor associated antigen (TAA), a receptor of a tissue cell, a receptor of a cancer cell, or a combination thereof.
In one embodiment, the NKG2D containing multi-specific antibody-like protein may mono-specific, bi-specific, tri-specific, tetra-specific or penta-specific.
In one embodiment, the NKG2D containing multi-specific antibody-like protein may have D2 comprising a dimer connected to CL and CH1, wherein the dimer is NKG2D. In one embodiment, the NKG2D containing mono-specific antibody-like protein may include an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identify to SEQ ID NO. 196 or 198.
In one embodiment, the NKG2D containing multi-specific antibody-like protein may have D1, D2, D3, D4, D5 and D6 each independently having a binding specificity against an antigen selected from EGFR, HER2, HER3, EGFRvIII, ROR1, CD3, CD28, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D, NKG2D ligand, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, PD-L1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40, CD40L, VISTA, ICOS, BTLA, LIGHT, HVEM, CSF1R, CD73, CD39, CLDN18.2, DLL3, HLA-G, FcRH5, GPRC5D, LIV-1, MUC1, CD138, CD70, CD16, uPAR, Siglec-15, CD47, CD38, NKp46, PD-L2, CD160, LOX-1, SIRPα, CD27, and wherein the Fc domain may comprise a human IgG Fc domain.
In one embodiment, the NKG2D containing multi-specific antibody-like protein may have D2, D5, and D6 each independently having a binding specificity against a tumor associated antigen. In one embodiment, the NKG2D containing multi-specific antibody-like protein may have D2 having a binding specificity against a tumor associated antigen. In one embodiment, the NKG2D containing multi-specific antibody-like protein may have D1, D2, D3, and D4 each independently having a binding specificity against NKG2D ligands, CD3, PD-L1, 4-1BB or a combination thereof.
In one embodiment, the NKG2D containing multi-specific antibody-like protein an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of sequence identify to SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 78, 80, 82, 84, 86, 88, 30 or 32.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against NKG2D ligands, D2 having a binding specificity against CD3, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against NKG2D ligands, D2 having a binding specificity against CD3, D3 having a binding specificity against 4-1BB, and D4 having a binding specificity against PD-L1.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against 4-1BB, D2 having a binding specificity against PD-L1, D3 having a binding specificity against CD3, and D4 having a binding specificity against NKG2D ligands.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against PD-L1, D2 having a binding specificity against 4-1BB, D3 having a binding specificity against CD3, and D4 having a binding specificity against NKG2D ligands.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against a tumor-associated antigen, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a specificity against NKG2D ligands.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against an antigen selected from a group consisting of mesothelin, claudin 18.2, HER2, EGFRvIII, and CD33, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D5 having a specificity against NKG2D ligands.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3 and D2 having a binding specificity against NKG2D ligands.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against NKG2D ligands, and D6 having a binding specificity against a tumor-associated antigen.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against NKG2D ligands, and D6 having a binding specificity against CD19.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against NKG2D ligands, D3 having a binding specificity against PD-L1, and D4 having a binding specificity against 4-1BB.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against NKG2D ligands, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D6 having a binding specificity against a tumor-associated antigen.
In one embodiment, the NKG2D containing multi-specific antibody-like protein D1 having a binding specificity against CD3, D2 having a binding specificity against NKG2D ligands, D3 having a binding specificity against PD-L1, D4 having a binding specificity against 4-1BB, and D6 having a binding specificity against CD19.
In a second aspect, the application provides isolated nucleic acid sequences encoding an amino acid sequence of the multi-specific antibody-like protein as disclosed thereof.
In a third aspect, the application provides expression vector. In one embodiment, the expression vector includes isolated nucleic acid sequence as disclosed herein.
In a further aspect, the application provides host cell including the isolated nucleic acid sequences disclosed herein. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is a eukaryotic cell.
In a further aspect, the application provides method for producing a multi-specific antibody-like proteins of their fragments thereof. In one embodiment, the method may include the steps of culturing a host cell may include an isolated nucleic acid sequence such that the DNA sequence encoding the multi-specific antibody or monomer is expressed, and purifying said multi-specific antibody, wherein the isolated nucleic acid sequence encodes an amino acid of the multi-specific antibody-like proteins or their fragments thereof.
In a further aspect, the application provides immunoconjugates. In one embodiment, the immuno-conjugate may include a cytotoxic agent or an imaging agent linked to the multi-specific antibody of claim 30 through a linker, wherein the linker may comprise an ester bond, an ether bond, an amid bond, a disulphide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphorus ester bond, a peptide bond, a hydrophobic poly(ethylene glycol) linker, or a combination thereof. In one embodiment, the cytotoxic agent or the imaging agent may comprise a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent from class of calicheamicin, an antimitotic agent, a toxin, a radioactive isotope, a toxin, a therapeutic agent, or a combination thereof.
In a further aspect, the application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable carrier and the multi-specific antibody-protein or their fragments thereof, the immuno-conjugate disclosed herein, or both. In one embodiment, the pharmaceutical composition further may include a therapeutic agent selected from a radioisotope, radionuclide, a toxin, a chemotherapeutic agent or a combination thereof.
In a further aspect, the application provides method for treating or preventing a cancer, an autoimmune disease, or an infectious disease in a subject. In one embodiment, the method includes the steps of administering a pharmaceutical composition that may include a purified multi-specific antibody-like protein of their fragments thereof. In one embodiment, the method may include administering to the subject an effective amount of the purified multi-specific antibody-like protein, immunoconjugates, or pharmaceutical composition disclosed herein.
In one embodiment, the method may further include co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent may comprise an antibody, a chemotherapy agent, an enzyme, an anti-estrogen agent, a receptor tyrosine kinase inhibitor, a kinase inhibitor, a cell cycle inhibitor, a check point inhibitor, a DNA, RNA or protein synthesis inhibitor, a RAS inhibitor, an inhibitor of PD1, PD-L1, CTLA4, 4-1BB, OX40, GITR, ICOS, LIGHT, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA, B7H4, CSF1R, NKG2D ligand, CD73, or a combination thereof.
In one embodiment, the subject is a human. In one embodiment, the subject is a mammal. In one embodiment, the subject is chimpanzee. In one embodiment, the subject is a pet animal.
In a further aspect, the application provides solutions including an effective concentration of the purified multi-specific antibody-like protein or their fragments thereof, immunoconjugates, or pharmaceutical composition as disclosed herein. In one embodiment, the solution is blood plasma in a human subject.
The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.
The disclosure provides, among others, isolated antibodies, methods of making such antibodies, bispecific or multi-specific molecules, antibody-drug conjugates and/or immuno-conjugates composed from such antibodies or antigen binding fragments, pharmaceutical compositions containing the antibodies, bispecific or multi-specific molecules, antibody-drug conjugates and/or immuno-conjugates, the methods for making the molecules and compositions, and the methods for treating cancer using the molecules and compositions disclosed herein.
The present application relates to methods of making and using multi-specific GNC antibodies, in particular, tetra-, penta-, and hexa-specific GNC (tetraGNC, pentaGNC, hexaGNC) antibodies. In general, GNC proteins, such as GNC antibodies, are characterized by comprising two moieties: moiety 1 for engaging immune cells, such as activating T cells, while moiety 2 targeting tumor cells. GNC antibodies retain multiple antigen binding domains for engaging immune cells, such as anti-CD3 for T cell activation, anti-4-1BB for co-stimulation, and anti-PD-L1 for inhibiting immune checkpoint. To improve the efficacy of antibody therapy for treating cancer, GNC antibodies are designed to be structurally stable and compact while retaining the characteristic feature of two moieties in GNC antibodies. This improvement allows an additional binding specificity to a second tumor associated antigen on the same or different tumor cell. GNC antibodies contain an Fc domain that allows for FcRn-mediated recycling and half-life extension, as well as facile protein A-based purification. The Fc receptor-mediated immunity may be incorporated if desired. GNC antibodies are usually larger than an IgG antibody due to increased number of antigen binding domains (AgBD), which provides spatial flexibility for binding to both a T cell and a tumor cell. GNC antibodies may be an efficacious antibody therapeutics for treating cancer by targeting one or more tumor antigens, including but not limited to, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, HER2, HER3, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2. Multi-specific T-cell engaging antibodies, such as tetra-GNC and penta-GNC antibodies, have distinctive advantages over conventional immunotherapies. They demonstrate cross-linking functionality of CD3 on T cells with tumor associated antigens (TAAs), which re-directs and guides them to kill tumor cells without the need of removing T cells from the patient and/or genetically modifying them to be specific for tumor cells before re-introducing them back into the patient (also known as chimeric antigen receptor T cells, or CAR-T therapy). A GNC protein-mediated antibody therapy or T cell therapy does not involve genetic modification of T cells, the latter of which may carry the risk of transforming modified T cells to clonal expansion, i.e. T cell leukemia.
The present application discloses tetra-, penta-, and hexa-specific GNC (tetraGNC, pentaGNC, hexaGNC) antibodies comprising a heavy chain (HC) and a light chain (LC) as elucidated in
The term “antibody” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments (e.g., Fab, F(ab′)2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, scFv, bispecific or bi-effective, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab′)2, scFv and Fv fragments, including the products of an Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above. In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically bind an antigen. The immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecule. In one embodiment, the antibody may be whole antibodies and any antigen-binding fragment derived from the whole antibodies. A typical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain is comprised of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus. Within the variable regions of the light and heavy chains there are binding regions that interacts with the antigen.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
The monoclonal antibodies may include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. 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) for a review) or from molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). In the present disclosure some antibodies were created by the immunization of rabbits with both human PD-L1 protein and cells transiently expressing human PD-L1 on the cell surface. Rabbits are known to create antibodies of high affinity, diversity and specificity (Weber et al. Exp. Mol. Med. 49:e305). B cells from immunized animals were cultured in vitro and screened for the production of anti-PD-L1 antibodies. Besides immunization of rabbits followed by B cell culture, other common strategies for antibody generation and discovery include immunization of other animals (e.g., mice) followed by hybridoma and/or display on phage, yeast, or mammalian cells; or display using synthetic variable gene libraries. The antibody variable genes were isolated using recombinant DNA techniques and the resulting antibodies were expressed recombinantly and further screened for desired features such as ability to inhibit the binding of PD-L1 to PD-1, the ability to bind to non-human primate PD-L1 and the ability to enhance human T-cell activation. This general method of antibody discovery is similar to that described in Seeber et al. PLOS One. 9:e86184 (2014).
The term “antigen- or epitope-binding portion or fragment” refers to fragments of an antibody that are capable of binding to an antigen. These fragments may be capable of the antigen-binding function and additional functions of the intact antibody. Examples of binding fragments include, but are not limited to a single-chain Fv fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody connected in a single polypeptide chain by a synthetic linker or a Fab fragment which is a monovalent fragment consisting of the VL, constant light (CL), VH and constant heavy 1 (CH1) domains. Antibody fragments are produced using conventional methods known to those skilled in the art. The antibody fragments are can be screened for utility using the same techniques employed with intact antibodies.
The “antigen- or epitope-binding fragments” can be derived from an antibody of the present disclosure by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, 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.
Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab” fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
The Fab fragment may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other, chemical couplings of antibody fragments are also known.
“Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody.
The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda (λ), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
A “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain “humanized antibodies” are well known to those skilled in the art. (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology, 9:421 (1991)).
The terms “polypeptide”, “peptide”, and “protein”, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by a peptide bond.
The terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.
By “isolated” is meant a biological molecule free from at least some of the components with which it naturally occurs. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic a binding specificity.
“Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells.
The term “antigen” refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.
Also, as used herein, the term “immunogenic” refers to substances which elicit or enhance the production of antibodies, T-cells or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells and other reactive immune cells against administered immunogenic compositions of the present disclosure to moderate or alleviate the disorder to be treated.
“Specific binding” or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.
The term “affinity” refers to a measure of the attraction between two polypeptides, such as antibody/antigen, receptor/ligand, etc. The intrinsic attraction between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) of a particular interaction. A KD binding affinity constant can be measured, e.g., by Bio-Layer Interferometry, where KD is the ratio of kdis (the dissociation rate constant) to kon (the association rate constant), as KD=kdis/kon.
Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10−4 M, at least about 10−5 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, alternatively at least about 10−10 M, at least about 10−11 M, at least about 10−12 M, or greater, where KD refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope.
Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.
“Homology” between two sequences is determined by sequence identity. If two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs. The deviations appearing in the comparison between a given sequence and the above-described sequences of the disclosure may be caused for instance by addition, deletion, substitution, insertion or recombination.
The present disclosure may be understood more readily by reference to the following detailed description of specific embodiments and examples included herein. Although the present disclosure has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.
DNA sequence encoding heavy and light chains was cloned into pTT5 vector from custom gene fragments using restriction cloning and/or Gibson assembly. Plasmid DNA was transfected transiently in ExpiCHO cells (Thermo A29133) according to manufacturer's instructions to generate multispecific GNC protein. Titers were measured after 7-9 days using ForteBio Octet instrument with protein A sensors.
GNC proteins were purified via protein A chromatography (Cytiva, 17549853), washing with phosphate-buffered saline (PBS), and eluting in 50 mM sodium acetate, pH 3.6, followed by immediate neutralization with 1/5 addition of 1M sodium acetate pH 7.0. Analytical size-exclusion chromatography (aSEC) was performed to assess protein quality following affinity purification. aSEC was performed using an Acquity Arc Waters with a XBridge BEH SEC 300 Å, 7.8×300 mm, 3.5 μm column. Proteins were further polished with a preparative SEC step using Superdex 200 Increase 10/300 GL column. All subsequent assays were performed with protein that was at least 90% protein of interest by aSEC.
T-cell dependent cytotoxicity (TDCC) assay is based on the methods outlined in Nazarian et al. (2014). Target cells from established cancer cell lines (ADCC) are first transduced with a luciferase expressing gene to generate luciferase positive target cells. These target cells are next grown in cell culture flasks and when an appropriate number have been expanded they are lifted, counted and replated into 384 well (Corning 3570) at an appropriate density depending on previous growth characteristics, using a BioTek EL406 liquid dispenser. For adherent cell lines the cells are allowed to adhere to the plates overnight in a CO2 controlled jacketed tissue culture incubator. Next, either PBMC or previously expanded T-cells (dynabeads) are plated at an appropriate effector to target ratio, frequently 5:1, and the plate is dosed with a dilution series of the test T-cell targeting agent. Test article experiments are conducted in quadruplicate as the 96 well dilution block is stamped into 384 well quadrants robotically (Opentrons OT-2 liquid handling robot). The TDCC assay plate is incubated for 72-96 hours. Readout of the cell viability curve is accomplished by the use of the Promega Bright-glo luciferase assay kit. Briefly, 20 uL are added to the TDCC assay plate and incubated for ˜15 min before measuring the resultant luminescence on a BMG Clariostar plate reader. Killing curves and EC50 values are analyzed and plotted in GraphPad Software.
Analytical size exclusion chromatography (SEC) in combination with multi-angle light scattering (MALS) and absorbance (UV) and/or refractive index (RI) concentration detectors. SEC-MALS is typically employed during analytical characterization and early phase clinical trials of antibody to support FDA IND submission. Our size exclusion is performed using an Acquity Arc Waters with a XBridge BEH SEC 300 Å, 7.8×300 mm, 3.5 μm column. The MALS component uses Wyatt miniDAWN TREOS/Optilab T-rEX systems. The molecular weight may be determined from a measurement of the change, Δn, of the solution's refractive index n with the molecular concentration change, Δc, by measuring the dn/dc (=Δn/Δc) value using an Optilab T-rEX differential refractometer. The intensity of light scattered by a molecule, measured by means of a miniDAWN TREOS multi-angle light scattering (MALS) detector, is directly proportional to the molar mass.
ForteBio Octet platform applies Bio-Layer Interferometry (BLI) as a label-free technology for measuring protein-protein interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. In this method, the binding between an antibody immobilized on the Anti-human IgG Fc Capture (AHC) Biosensors tip surface and an antigen in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, Δλ, directly reflecting the change in thickness of the biological layer. The interaction of these two molecules are measured in real time, providing the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate do not affect the interference pattern. Facile analysis of the binding of GNC proteins to targeted antigens can be performed on the Octet system, using AHC tips to immobilize the GNC protein, and using purified antigen as analyte either as a single concentration (e.g., 100 nM) or a series of concentrations (e.g., seven 1:2 serial dilutions starting at 200 nM).
The hydrodynamic radius (Rh) of antibody samples (1 mg/ml) was measured by using a DynaPro Plate Reader (Wyatt Technology, Santa Barbara, Calif.) from 25° C. to 75° C. in 1° C. increments with ramp rate of 0.26° C./min. A total of 3 acquisitions of 5 s were collected at each temperature. DYNAMICS 7.8.1.3 software (Wyatt Technologies) was used to calculate the radius and the onset temperature at which Rh begins to change significantly and the midpoint of the transition curve (Tm).
Quantitative Flow Cytometry (qFACS)
CountBright absolute counting beads (Thermo C36950) were used for calibration, and primary antibodies panitumumab (EGFR), anti-HER3 from MM111 (HER3), PL221G5 (PD-L1), TF 3H8-1 (CEA), and trastuzumab (HER2) were used to quantify the corresponding receptor number on various tumor cell lines, as described previously (Wang L et al. Curr Protoc. Cytom. 2016).
NKG2D is a major recognition receptor for the detection and elimination of transformed and infected cells as its ligands are induced during cellular stress, either as a result of infection or genomic stress such as in cancer. In humans, NKG2D is encoded by KLRK1 gene which is located in the NK-gene complex (NKC), it is expressed by NK cells, γδ T cells, and CD8+ αβ T cells. Human NKG2D receptor complex assembles into a hexameric structure, while NKG2D itself forms a homodimer whose ectodomains serve for ligand binding. In NK cells, NKG2D serves as an activating receptor, which itself is able to trigger cytotoxicity. The function of NKG2D on CD8+ T cells is to send co-stimulatory signals to activate them. The major histocompatibility complex class I polypeptide-related sequence A gene (MICA) encodes a membrane-bound protein acting as a ligand to stimulate an activating receptor, NKG2D, expressed on the surface of essentially all human natural killer (NK), γδ T and CD8+ αβ T cells. MICA protein is absent from most cells but can be induced by infections and oncogenic transformation and is frequently expressed in epithelial tumors. Upon binding to MICA, NKG2D activates cytolytic responses of NK and γδ T cells against infected and tumor cells expressing MICA. Therefore, membrane-bound MICA acts as a signal during the early immune response against infection or spontaneously arising tumors. On the other hand, human tumor cells spontaneously release a soluble form of MICA, causing the downregulation of NKG2D and in turn severe impairment of the antitumor immune response of NK and CD8+ T cells. This is considered to promote tumor immune evasion and also to compromise host resistance to infections, which may be neutralized by free NKG2D. In this context and by the definition of GNC proteins, NKG2D is one of cytotoxic cell binding moieties (from Applicant's application No. PCT/US2018/039160, incorporated herein in its entirety).
The GNC antibodies having NKG2D ligand binding specificity comprise a common core antibody domain, the Fc region of which can either have or be devoid of effector function. The tetraGNC antibodies having NKG2D dimer as one of binding domain on the heavy chain (HC) have been generated (Table 1). These antibodies had either 2 additional scFv domains, including the binding domain to 4-1BB, a TNF superfamily receptor normally expressed on activated T cells (SI-49E1, SI-49E2, SI-49E3, SI-49E4, Table 1), or 1 additional scFv plus 41BBL (trimer form), which is normally found on antigen-presenting cells (APCs) and binds to 4-1BB (SI-49E11, SI-49E12, SI-49E13, Table 1). The four binding domains (D1 through D4) were fused through G/S linker regions and expressed as a single heavy chain (HC or Chain A or Chain 1). By definition as shown in
Analytical SEC shows the stability and high quality of purified tetraGNC antibodies comprising NKG2D receptor and 41BBL (
In NK cells, NKG2D serves as an activating receptor, which itself can trigger cytotoxicity, whereas on CD8+ T cells the function of NKG2D is to send co-stimulatory signals to activate them. NKG2D forms a homodimer whose ectodomains serve for ligand binding. This feature qualifies NKG2D as a non-variable-sequence-based binding domain in a GNC format and other binding domains can be added to create a class of multispecific NKG2D-GNC protein. In one GNC format, individual NKG2D monomer was incorporated in the D2 position on heavy chain and light chain which formed a dimeric NKG2D receptor on HC/LC dimerization. Thus, NKG2D can act as a receptor for the multi-specific antibody-like protein GNC molecule to bind its ligand. In other GNC format, a NKG2D tandem repeat was designed by adding a (GxSy)n linker between individual NKG2D monomers which homodimerizes and forms a functional dimeric receptor. This NKG2D tandem dimeric structure can be positioned in D1, D3, D4, D5, or D6.
To evaluate the effect of NKG2D position in tetraGNC antibodies, SI-49E1, SI-49E2, SI-49E3, SI-49E4 were used for TDCC assay (
To evaluate the effect of NKG2D position in pentaGNC antibodies, SI-49P1, SI-49P2, SI-49P3, SI-49P4, and SI-49P5 (Table 2) were used for TDCC assay (
To quantify the ability of NKG2D receptor domain dimer to redirect T cells to kill MICA-bearing tumor cells in the context of tetraGNC or pentaGNC antibodies, TDCC assay with MDA-MB-231 target cells was performed. Test articles included a control tetraGNC (SI-38E72, αCD3×αFITC×αPD-L1×α4-1BB) with negative control anti-FITC domain at D2, tetraGNC antibodies (SI-49E1, SI-49E2, SI-49E3, SI-49E4, NKG2D×αCD3×αPD-L1×α4-1BB) with binding domains at different GNC positions, and pentaGNC (SI-49P1, αCD3×αMSLN×αPD-L1×α4-1BB×NKG2D) with additional αMSLN binding domain. The effector:target cell ratio (E:T) was 5:1, and the purified T cells, target cells, and drug dilutions were incubated for 96 hours before reading luminescence, representative of remaining tumor cells. Note that some experiments were performed on different days, and the absolute EC50 value may vary. Nevertheless, the results in Table 4 show that all tetraGNC antibodies containing the NKG2D domain had significantly higher potency of TDCC than the corresponding tetraGNC without NKG2D (SI-38E72), ranged from about 10-fold (SI-49E2) to more than 130-fold (SI-49E4). While the differences among these tetraGNC antibodies may be attributed to configuration of the same group of 4 bind domains, the addition of anti-TAA moiety significantly increased the potency of SI-49P1, a pentaGNC antibody, by more than 600-fold. Thus, adding one cytotoxic binding moiety, such as NKG2D, may improve the potency, and adding an anti-TAA binding moiety can specifically increase potency of T cell-mediated tumor cell killing.
Antibody-based proteins are most often purified via protein A affinity chromatography, where the protein A resin captures the antibody at a binding site at the CH2-CH3 interface in the Fc domain. However, protein A also binds to the VH domain domain of VH3 family Fvs. For most antibody-based platforms this is not a problem, since VH domains are generally on the heavy chain. However, when scFvs containing VH3 are attached to the light chain, the VH domain can bind to protein A resin during purification, causing light chain monomers and dimers to contaminate the desired heavy-light chain heterotetramer. Thus, a potential hurdle when producing multi-specific antibodies containing any VH3 domain on the light chain is the presence of additional contaminants in the protein A elution. This is especially problematic when the light chain expresses more efficiently than the heavy chain, causing an abundance of light chain contaminants to be purified along with the desired protein assembly.
In order to rationally disrupt protein A binding of VH3 family members, a structural approach was taken to interrupt the binding interface. Crystal structure 1DEE (Graille M. et al. Proc. Nat. Acad. Sci. 2000.) showed that residue R19 in VH3 (Kabat numbering) is in direct contact with two side chains of protein A domain D. In particular, contact with Q32 and D36 could be eliminated to significantly weaken the interaction. Thus, R19 was mutated to serine, which does not form these interactions due to its shorter side-chain. Additionally, S19 exists naturally in other VH family members, suggesting that it may be less immunogenic than other substitutions. The mutation R19S (Kabat numbering) was incorporated into the FR1 region of the VH domain for VH3-containing scFvs on the GNC light chain. Specifically, the hexa-GNC antibody, SI-55-H11, harbored R19S mutation in its light chain sequences encoding the anti-HER3 scFv domain at D5 and the anti-CD19 scFv at domain 6. The residue of interest is at the protein A binding interface, and therefore mutation of R to S disrupts the interaction with protein A. Elimination of protein A binding in light chain scFvs prevents light chain monomers and dimers from binding to protein A during purification. As a result, a more homogeneous product without light chain contaminants can be obtained. For hexa-GNC, which may contain up to two VH3 scFvs per light chain, this mutation is especially important in allowing efficient purification of the desired product.
In a general scheme of multi-specific GNC proteins (
The TDCC assay was set under the same conditions, such as the Effector:Target cell ratio (E:T) was 5:1, and the purified T cells, target cells, and drug dilutions were incubated for 72 hours before reading luminescence, representative of remaining tumor cells. Note that some experiments were performed on different days, the EC50 values may vary from one experiment to the other within an error margin. Nevertheless, the potency of these pentaGNC and hexaGNC antibodies were under 1 pM and within a ten-fold range, indicating the structural variations may improve manufacturing cost and feasibility more significantly than the efficacy of killing BXPC3 cells. In this context, the composition of binding specificity remains to be the determining factor for creating multi-specific GNC antibodies targeting specific forms of cancer.
With the capacity of up to six binding specificities, multi-specific GNC antibodies may become antibody therapy with the highest potency of killing cancer cells, for example, the value of EC50 may be down to the range of pM or even fM from nM. A successful and highly efficacious multi-specific GNC antibody depends on the composition of both moiety 1 and moiety 2 antigens. Table 4 established the comparative potency of four moiety 1 binding specificities (i.e. αCD3, αPD-L1, α4-1BB, and NKG2D) in tetraGNC antibodies in TDCC assay using MDA-MB-231 cells as target breast cancer cells. As compared to the control antibody (SI-38E72) comprising a trio of moiety 1 binding domains (i.e. αCD3, αPD-L1, and α4-1BB as D1, D3, and D4 of HC, respectively), the addition of fourth moiety 1 binding domain, a non-variable sequence-based NKG2D dimer receptor improved the potency by about 10- to 130-fold depending the configurations. However, the addition of an anti-TAA moiety significantly increased the potency of the pentaGNC antibody (SI-55H11) up to 600-fold.
To evaluate the moiety 1 binding specificity in combination with the trio moiety 1 binding domains, TDCC assay was performed using the breast cancer cell line MDA-MB-231 as target cells. All test articles included αCD3, αPD-L1, and α-4-1BB scFvs at positions D1, D3 and D4, respectively. The tetraGNC antibody (SI-38E72) was used as a control for the absence of moiety 1 binding domain, with α-FITC domain at D2 which is not specific for any tumor antigen. Other tetraGNC test articles contained various binding domains at D2 (SI-55E: αEGFR Cetuximab; SI-55E2: αEGFR Panitumumab; SI-50E1: αHER2 Trastuzumab; and SI-51E1: αMesothelin Amatuximab), and the pentaGNC antibody (SI-1P1) contained αEGFR Cetuximab at D2 and αHER3 MM111 scFv at D5. The effector:target cell ratio (E:T) was 5:1 or 10:1, and the purified T cells, target cells, and drug dilutions were incubated for 96 hours before reading luminescence, representative of remaining tumor cells. Note that some experiments were performed on different days, the EC50 values might vary but were within an error margin. Nevertheless, the results demonstrate that all GNC antibodies containing an αTAA domain at D2 elicited significantly (20- to 100-fold) more potent TDCC than the control with αFITC at D2 (Table 6). The EC50 for the pentaGNC antibody (SI-1P1) was similar to that for the tetraGNCs, indicating that domains can be added to increase TAA selectivity while still retaining potent TDCC.
The configuration of the trio of moiety 1 binding domains being fixed at D1, D3, and D4 (Table 6) may be used as a backbone HC to accurately identify new and/or effective moiety 2 binding domains of TAAs. To assess the ability of multi-specific GNC antibodies targeting different tumor antigens to elicit T cell-mediated killing, TDCC assay was performed using the cervical cancer cell line HeLa as target cells. All test articles included αCD3, αPD-L1, and α4-1BB scFvs at positions D1, D3 and D4, respectively, and tetraGNC antibody (SI-38E72) was used as a control for having αFITC domain at D2 which is not specific for any tumor antigen. Other tetraGNC test articles contained various binding domains at D2 as listed in Table 7. The effector:target cell ratio (E:T) was 10:1, and the purified T cells, target cells, and drug dilutions were incubated for 96 hours before reading luminescence, representative of remaining tumor cells. Note that some experiments were performed on different days, so that the EC50 values may vary but were in an error margin. Nevertheless, the result from the same day experiment show that the two anti-EGFR tetraGNC antibodies, SI-55E1 (Cet) and SI-55E2 (Pan), killed more than 50% of cancer cells with a potency at 13 and 9 pM, respectively (
To further characterize the comparative potency of SI-55E1 and SI-55E2 having the same configuration of moiety 1 binding domains and targeting EGFR, MDA-MB-231 cell line was used in TDCC assay. In the same day experiment, two additional antibodies targeting both EGFR and HER3 were used: SI-1P2 is a pentaGNC antibody having the same configuration of moiety 1 binding domains as that of SI-55E1 and SI-55E2 plus an additional moiety 2 domain binding to HER3, and SI-1 is a bispecific antibody against both EGFR and HER3 in the absence of any moiety 1 binding domain. The materials and methods of this TDCC assay were the same as described in Example 1. As shown in
To measure the added effect of moiety 1 binding domain, such as anti-CD3, two tetraGNC antibodies (SI-50E1 and SI-50E6) and one biGNC antibody (SI-50X1) were analyzed in TDCC assay using MDA-MB-231 cell line as target cells. All three antibodies have the same binding specificity to HER2 derived from Trastuzumab (Table 6). SI-50E1 and SI-50E6 have the same configuration of moiety 1 and moiety 2 binding domains, however, the scFv domains of SI-50E6 were engineered with additional disulfide bonds for added stability (i.e. stapled) whereas the scFv domains of SI-50E1 was not stapled. SI-50X1 is a bispecific antibody targeting CD3 and HER2. The TDCC dose-response curves clearly showed that all three GNC antibodies are potent and their EC 50 values were in the range of fM (
Antibody therapy employs a wide variety of strategy to kill cancer cells either directly or indirectly, and both mechanisms of action depend on binding to surface antigen(s). On the other hand, the cancer cells are evolved to gain their ability of escaping such recognitions, either from the antibody, immune cells, or both. With the capacity of up to six binding specificities, multi-specific GNC antibodies displayed the highest potency as measured by the in vitro EC50 in the range of pM and fM. A highly efficacious multi-specific GNC antibody depends on the composition of both moiety 1 and moiety 2 antigens. Herein, the trio of moiety 1 binding domains (CD3, PD-L1, and 4-1BB) in a configuration (D1, D3, and D4 of HC, respectively) provides the backbone of multi-specific GNC antibodies. Such formatted GNC antibodies allow selecting, screening, and optimizing TAAs of target cancer cells (
To demonstrate the importance of TAA surface expression profile, quantitative flow cytometry (qFACS) was performed to quantify the approximate number of receptors per cell for various tumor targets. EGFR, HER2, and HER3 are members of the EGFR family whose expression is often upregulated by solid tumors, and PD-L1 is the target for inhibiting the immune checkpoint signaling utilized by a portion of human cancer. Nonetheless, the surface expression of HER3 and PD-L1 was not detectable in MDA-MB-232 cells and Hela cells, respectively (Table 8). The observation may provide an explanation for lacking a synthetic lethal effect when targeting both EGFR and HER3 by SI-1P1 as compared to SI-55E1, SI-55E2, and SI-50E1 (Table 6), each of which targets either EGFR or HER2. It may also explain that the failure of inducing TDCC by control antibody (SI-38E72) is likely due to the absence of PD-L1 on Hela cells (Table 7). In light of evolving and dynamic expression profile of cancer cells, any candidate antibody may be tested for TDCC using different types of cancer cells, such as those shown in Table 9.
To screen for TAAs, a modular cloning platform may be used to efficiently identify TAAs or epitopes of TAAs for assembling moiety 2 binding domains for multi-specific GNC antibodies. For example, TAA-Fc tetraGNC-1 and TAA-Fc tetraGNC-2 are two groups of tetraGNC antibodies with the pair-wise identical binding specificities. The only difference is that all TAA-Fc tetraGNC-2 antibodies have stapled scFv domain D1, D3, and D4 (mutations of VH 44->C, and VL 100->C) of HC. In this case, HC was swapped to produce two groups of tetraGNC antibodies. In another examples, LC may be swapped to produce multi-specific GNC antibodies with an added binding domain to TAA, such as SI-55P10 to SI-55H11, and SI-55E1 to SI-1P1. This modular cloning platform allows efficient assembly of multi-specific GNC antibodies with up to 3 TAAs starting from a single anti-TAA monoclonal antibody.
To further validate the flexibility of the GNC platform to accommodate diverse binding domains in each molecular position, a panel of proteins was generated with the NKG2D receptor at the D2 position (Table 10). When monomeric NKG2D is incorporated into the D2 position of both GNC chains, it was predicted that the NKG2D monomers would dimerize upon chain association. SI-49R21 is a monospecific GNC (antibody-like protein) with NKG2D receptor in place of the antibody VH/VL domains. SI-49R22 contains the same format, except that its Fc domain also contains knobs-into-holes mutations (chain A: T366S/L368A/Y407V; and chain B: T366W) for heterodimerization. SI-49R23 is a monospecific protein with NKG2D directly fused to the antibody Fc domain, and SI-49R24 contains this same structure but additionally has knobs-into-holes mutations in the Fc. SI-49R19 is a bispecific GNC with anti-CD3 scFv in D1 and NKG2D in D2, and SI-49R18 is a tri-specific GNC which additionally contains anti-CD19 at D6. SI-49E15 contains anti-CD3 scFv at D1, NKG2D at D2, anti-PD-L1 scFv at D3, and anti-4-1BB scFv at D4; SI-49P6 contains the same domains as SI-49E15, and additionally contains anti-CD19 scFv at D6. SI-49P7 has the same structure as SI-49P6, except it contains 4-1BB ligand trimer at D4 instead of anti 1BB scFv.
All the proteins with NKG2D in D2 were successfully expressed and purified. To validate functionality of the NKG2D receptor in the D2 position, Octet binding was performed. GNC protein was loaded onto AHC sensors at 5 ug/ml, and subsequently bound to 1:2 serial dilutions (top concentration 100 nM) of human MICA (Acro, MIA-H5221). The binding affinities (KD values) resulting from global fit to a 1:1 binding model are shown in Table 11. Mono-, bi-, tri-, tetra-, and penta-GNC proteins with NKG2D at D2 were shown to retain strong binding to the NKG2D ligand MICA, as demonstrated by KD values under 20 nM.
To further demonstrate the adaptability of the GNC platform, a panel hexa-specific GNC proteins targeting the same antigens (CD3×EGFR×PD-L1×4-1BB×CD19×HER3) was generated. The entire panel contained anti-PD-L1 scFv at D3, anti-4-1BB scFv at D4, anti-HER3 scFv at D5, and anti-CD19 scFv at D6. Two molecules (SI-77H4 and SI-77H5) contained an anti-CD3 scFv at D1 and anti-EGFR Fab at D2, the difference being that the anti-EGFR domain for SI-77H4 was humanized while that of SI-77H5 retained mouse sequences. Two molecules (SI-55H11 and SI-55H12) contained anti-EGFR scFv at D1 and anti-CD3 Fab at D2, the difference being that the D2 VH/VL contained a disulfide staple in SI-55H11 (VH-44C, VL-100C) but not in SI-55H12. Thus, the panel allowed for clarification of whether D1/D2 positioning affected either protein expression properties or binding affinity to the targeted tumor-associated antigen.
Proteins were expressed transiently in ExpiCHO cells as described in Example 1. After approximately 8 days, GNC titers were measured using protein A sensors on the Octet platform (Table 12). Results demonstrate that the hexa-specific GNC proteins expressed well (≥30 μg/ml) regardless of the positioning and format of the anti-EGFR and anti-CD3 domains. After the first protein A purification step, all proteins had similarly low levels of aggregation with the percent protein of interest ranging from 72 to 85% (Table 12). Next, the affinity of the anti-EGFR domains for human EGFR was assessed by loading GNC proteins onto AHC sensors and using a single concentration (100 nM) of His-tagged human EGFR (expressed in-house) as the analyte. As shown in Table 12, the positioning and format of anti-EGFR and anti-CD3 domains did not significantly affect EGFR binding affinity (KD values within approximately 2-fold). Thus, GNC proteins retain full functionality regardless of the positioning of anti-TAA and anti-CD3 domains between D1 and D2.
a284A10, see Applicant's application No. PCT/US2018/039143;
bSI-huBU12, see Applicant's application No. PCT/US2020/059230.
GYSWGNVDNVFGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKGLEY
VGGAFGGGTKVEIKGGGGSGGGGSGEVQLLESGGGLVQPGGSLRLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACI
AAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGG
AAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSASTKGPS
RDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSGGGGSGGGGS
AFSFDYAMDLWGQGTLVTVSTGGGGSGGGGSRSLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKGLEY
DITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSGGGGSGGGGSG
GYSWGNVDNVFGGGTKVEIKGGGGSGGGGSGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA
AFSFDYAMDLWGQGTLVTVSTGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
SYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDL
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
SYWMSWVRQAPGKCLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDL
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKG
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
SYWMSWVRQAPGKCLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDL
GSSAITSNNIWGQGTLVTVSSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKL
AGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGG
DSSSYYMFNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSALVMTQSPSTLSASVGDRVTINCQASEDIDTYLAWY
SYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDL
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLE
IAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSG
DSGYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQ
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKL
AGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGG
GYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKP
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLE
AGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGG
GYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKP
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
YDMCWVRQAPGKCLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAM
DLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAP
YVGGAFGCGTKVEIK
TSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYF
DYWGQGTLVTVSSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
YDMCWVRQAPGKCLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAM
DLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAP
TSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYF
DYWGQGTLVTVSSGGGGSGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
YDMCWVRQAPGKCLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAM
DLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAP
YVGGAFGCGTKVEIK
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE
AGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSG
DPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKA
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE
AGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGSG
DPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKA
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLE
IAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSG
DSGYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQ
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLE
IAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSG
DSGYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQ
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKG
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKG
CIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGS
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLE
SWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGSGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGGGTKVEI
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLE
SWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGSGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGCGTKVEI
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLE
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLE
WIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSAASTKGP
AAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGG
SGYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQK
GSSAITSNNIWGQGTLVTVSTGGGGSGGGGSQIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLE
WIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSAASTKGP
AAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGG
SGYSDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQK
GRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSASTKGPSVF
SAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAMDLWGQGTLVTVSSGGGGSGGGGS
SDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGK
GVGYFDLWGRGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIY
EASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPS
HIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGG
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
SAITSNNIWGQGTLVTVSTGGGGSGGGGSFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQA
YDMCWVRQAPGKCLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSAFSFDYAM
DLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSISSHLNWYQQKPGKAP
YVGGAFGCGTKVEIK
This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 62/991,042 filed Mar. 17, 2020 under 35 U.S.C. 119(e), the entire disclosures of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/22849 | 3/17/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62991042 | Mar 2020 | US |
