Patent Application
20230002488
The present application relates to the technical field of multi-specific antibody for immunotherapy and more particularly relates to making and using Guidance and Navigation Control (GNC) antibodies with multiple binding activities against surface molecules of immune cells and tumor cells.
Cancer develop by gaining mutations that enable the cancer cells to transform, proliferate, and metastasize while escaping from the immune surveillance and response. Antibody therapy for treating cancer recruits multiple distinct mechanisms. For example, monoclonal antibodies targeting growth receptors (EGFR, HER2, etc.) that are overexpressed on tumor cells can be used to block tumor cell proliferation. Using antibodies to block inhibitory T cell checkpoint signals (anti-PDL1, anti-PD1, anti-CTLA4) is a strategy to prevent tumor cells from weakening the immune response that would otherwise seek to control their growth. Another therapeutic strategy is to inhibit angiogenesis (e.g., anti-VEGF), where the reduced access to oxygen and nutrients slows the growth of tumor cells. Monoclonal antibodies and antibody-drug conjugates (ADCs) are initially effective at controlling tumors. However, cancer resistance to antibody therapy often occurs through escape mechanisms, such as ectodomain shedding, receptor downregulation and receptor mutation (Miller et al. Clin Cancer Res. 2017; Reslan et al. Mabs. 2009; Loganzo et al. Mol Cancer Ther. 2016). For example, resistance to anti-HER2 mAb trastuzumab may occur through ectodomain shedding of HER2 or through occlusion of the trastuzumab epitope on HER2 (Fiszman and Jasnis. International Journal of Breast Cancer, 2011).
Combinational therapies combining multiple therapeutic mechanisms including that of chemotherapy, radiation therapy and antibody therapy have become a mainstream therapeutic strategy. In this context, multi-specific antibodies combine different antibody therapies and mechanisms into a single agent (Boumandi and de Sauvage. Nat Rev Drug Discov. 2020).
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.
In one aspect, the application provides guidance and navigation control (GNC) proteins that can simultaneously bind effector cells and target cells. The GNC protein may be a monomer or a dimer of the monomer. The GNC protein may be an antibody or an antibody-like protein. The GNC protein may have at least 5 or at least 6 binding domains.
In one embodiment, the application provides multi-specific antibody-like proteins having a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, a first binding domain (D1) at the N-terminal, a second binding domain (D2) comprising a light chain moiety, a Fc region, a third binding domain (D3), and a fourth binding domain (D4) at the C-terminal. The light chain moiety comprises a fifth binding domain (D5) covalently attached to the C-terminal, a sixth binding domain (D6) covalently attached to the N-terminal, or both. The D1, D2, D3, D4, D5 and D6 each has a binding specificity to a tumor antigen, an immune signaling antigen, or a combination thereof.
The tumor antigen may be a tissue antigen, a neoantigen, a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), or a combination there.
The D2 may include CH1. In one embodiment, the light chain moiety in the D2 may include CL. In one embodiment, the light chain moiety may include Cκ/Cλ.
The D2 may include a dimer.
In on embodiment, the D2 may include a Fab region. In one embodiment, the Fab region may have a disulfide bond between VL and VH. In one embodiment, the D2 may include a VL and a VH.
In one embodiment, the D2 may include a receptor. In one embodiment, the receptor may be NKG2D. In one embodiment, the D2 may include NKG2D connected to CH1 and CL. In one embodiment, the D2 may have an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 155 and 116.
The D2 may be connected to the Fc region through a hinge.
The Fc region may include null mutation, which may have the effect to reduce or eliminate effector functions. In one embodiment, the Fc region may be wild-type Fc. In one embodiment, the Fc region may include LALAKA mutations for null Fc. In one embodiment, the LALAKA mutations for null Fc may include L234A/L235A/K322A (Eu numbering) mutations. In one embodiment, the Fc region may include G237A (Eu numbering) mutation. In one embodiment, the Fc region may include N297A (Eu numbering) mutation. In one embodiment, the Fc region may include a glycosylated Fc. In one embodiment, the Fc region may be an aglycosylated Fc to reduce effector function.
In one embodiment, the application may provide a multi-specific antibody-like protein having a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, a first binding domain (D1) at the N-terminal, a second binding domain (D2) comprising a dimer connected to CL and CH1, a Fc region comprising CH2 and CH3, wherein the CH2 is connected to the CH1 through a hinge, a third binding domain (D3), and a fourth binding domain (D4) at the C-terminal. The light chain moiety may have a fifth binding domain (D5) covalently attached to the C-terminal, a sixth binding domain (D6) covalently attached to the N-terminal, or both. The D1, D2, D3, D4, D5 and D6 each may have a binding specificity to a tumor antigen, an immune signaling antigen, or a combination thereof.
The dimer in the D2 may include VL and VH pair connected to CL and CH respectively, in which case the D2 domain may be a Fab region, and the GNC protein may be a multi-specific antibody monomer or a multi-specific antibody.
In one embodiment, the multi-specific antibody-like protein may be either penta-specific or hexa-specific.
In one embodiment, the light chain moiety in the D2 may have a fifth binding domain (D5) covalently attached to the C-terminal, and the multi-specific antibody-like protein is penta-specific. In one embodiment, the light chain moiety may have a sixth binding domain (D6) covalently attached to the N-terminal, and the multi-specific antibody-like protein is penta-specific. In one embodiment, the light chain moiety may have a fifth binding domain (D5) covalently attached to the C-terminal and a sixth binding domain (D6) covalently attached to the N-terminal simultaneously, which makes the multi-specific antibody-like protein to be hexa-specific.
The D1, D2, D3, D4, D5, and D6 may be independently a scFv domain, a receptor, or a ligand.
The scFv domain may have the configuration of VLVH or VHVL from the N terminal to the C terminal. In one embodiment, the scFv domain may include R19S (Kabat) mutation. In one embodiment, the scFv domain may include a disulphide bond between VL and VH. In one embodiment, the disulfide bond may be between vL100 and vH44 (Kabat) of the scFv domain. In one embodiment, the scFv domain may have an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 72-112.
In one embodiment, the D1, D2, D3, D4, D5, and D6 may all be scFv domains.
In one embodiment, the D1, D2, D3, D4, D5 and D6 each may be independently a receptor or a ligand. In one embodiment, at least one, two, three, four, or five of the D1, D2, D3, D4, D5, and D6 may be a receptor or a ligand. In one embodiment, the D1, D2 D3, D4, D5, and D6 may all be receptors or ligands. In one embodiment, the D4, D5 or D6 may be a receptor or a ligand. In one embodiment, the receptor or a ligand may have an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 113-116.
In one embodiment, the D2 has a binding specificity to CD3 or a tumor associated antigen (TAA).
In one embodiment, the D1, D2, D3, D4, D5, and D6 independently has a binding specificity to an antigen selected from a receptor on a T cell, an immune checkpoint receptor, a co-stimulation receptor, a receptor of a lymphocyte or a myeloid cell, a tumor associated antigen (TAA), a tissue antigen, a neoantigen, a tumor-specific antigen (TSA), a glycoprotein, or a combination thereof.
In one embodiment, the binding domain for the receptor on the T cell may be adjacent to the binding domain for the tumor associated antigen (TAA). In one embodiment, the binding domain for the receptor on the T cell is adjacent to the binding domain for the receptor of a lymphocyte or a myeloid cell.
In one embodiment, the receptor on the T cell may be CD3, T cell receptor, or a complex thereof. In one embodiment, the immune checkpoint receptor may be PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, CD160, LOX-1, siglec-15, CD47, SIRPα, or a combination thereof. In one embodiment, the co-stimulating receptor may be 4-1BB, CD28, OX40, GITR, CD40, ICOS, CD27, CD30, CD226, or a combination thereof. In one embodiment, the tumor associated antigen (TAA) may be EGFR, HER2, HER3, HER4, EGRFVIII, CD19, claudin 18.2, BCMA, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, cMET, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TACI, TROP2, NKG2D ligands, PD-L1, or a combination thereof.
In one embodiment, the D1, D2, D3, D4, D5 and D6 each independently may have a binding specificity to an antigen selected from EGFR, HER2, HER3, EGFRvIII, ROR1, CD3, CD28, CEA, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, NKG2D ligands, 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, CLDN18.2, CSF1R, and wherein the Fc region comprises a human IgG Fc region.
In one embodiment, the D2 and D5 each independently has a binding specificity to a tumor associated antigen, a neoantigen, or a tumor-specific antigen (TSA).
In one embodiment, the D1 has a binding specificity to CD3, CD20, EGFR, or their derivative thereof. In one embodiment, the D2 has the binding specificity to EGFR, CD3, HER2, MSLN, NKG2D ligands, or their derivative thereof. In one embodiment, the D3 has a binding specificity to PD-L1. In one embodiment, the D4 may include a 4-1BBL trimer or has a binding specificity to 4-1BB or its derivative thereof. In one embodiment, the D5 has a binding specificity to HER3, CD19, NKG2D ligands, or their derivative thereof. In one embodiment, the D6 has a binding specificity to CD19.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 has a binding specificity to EGFR, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D5 has a binding specificity to HER3. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 1-8.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD20, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 9-12.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD20, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D5 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 13-16.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 has a binding specificity to MSLN, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D5 has a binding specificity to NKG2D ligands. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 17-20.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 has a binding specificity to HER2, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D5 has a binding specificity to NKG2D ligands. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 21-24.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 25-28.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 comprises 4-1BB ligand trimer, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 29-32.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 33-36.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 comprises 4-1BB ligand trimer, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 37-40.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 has a binding specificity to EGFR, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 41-44.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 45-48.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 comprises 4-1BB ligand trimer, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 49-52.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 comprises NKG2D, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, D6 has the binding specificity to EGFR. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 117-120.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 comprises NKG2D, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, D6 has the binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 123-126.
In one embodiment, the multi-specific antibody-like protein is penta-specific, and wherein the D1 has a binding specificity to CD3, D2 comprises NKG2D, D3 has a binding specificity to PD-L1, D4 comprises 4-1BB ligand trimer, D6 has the binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 127-130.
In one embodiment, the multi-specific antibody-like protein is hexa-specific, and wherein the D1 has a binding specificity to EGFR, D2 has a binding specificity to CD3, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, D5 has the binding specificity to HER3, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 53-60.
In one embodiment, the multi-specific antibody-like protein is hexa-specific, and wherein the D1 has a binding specificity to CD3, D2 has a binding specificity to EGFR, D3 has a binding specificity to PD-L1, D4 has a binding specificity to 4-1BB, D5 has the binding specificity to HER3, and D6 has a binding specificity to CD19. In one embodiment, the multi-specific antibody-like protein has an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of sequence identity to SEQ ID NO. 61-68.
In one embodiment, the D1, D3, D4, D5 or D6 may include a (GxSy)n linker. n may be an integer from 1 to 10. x may be an integer from 1 to 10. y may be an integer from 1 to 10.
In one embodiment, the application may provide a guidance navigation control (GNC) protein that include the multi-specific antibody-like protein as described thereof. In one embodiment, such GNC protein may be a dimer of the multi-specific antibody-like protein as described herein.
In one aspect, the application provides isolated nucleic acid sequences encoding an amino acid sequence of the multi-specific antibody-like protein or its fragments or derivatives as disclosed herein.
In one aspect, the application provides expression vector including the isolated nucleic acid sequence as described herein.
In one aspect, the application provides host cells comprising the isolated nucleic acid sequence as disclosed thereof. In one embodiment, the host cell may be a prokaryotic cell or a eukaryotic cell.
In one aspect, the application provides methods for producing GNC proteins as disclosed herein. In one embodiment, the method for producing a multi-specific antibody or monomer as disclosed herein may include the steps of culturing a host cell comprising 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 protein as disclosed herein.
In one aspect, the application provides immuno-conjugate comprising a cytotoxic agent or an imaging agent linked to the GNC protein such as a multi-specific antibody-like protein or a multi-specific antibody disclosed herein through a linker. The linker may include a covalent bond such as 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 be 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 one aspect, the application provides pharmaceutical composition for treating, preventing or controlling conditions such as cancer, autoimmune diseases, or infectious diseases. In one embodiment, the composition may include a pharmaceutically acceptable carrier and a GNC protein such as a multi-specific antibody or a multi-specific antibody-like protein, their immuno-conjugate or their fragment thereof.
In one embodiment, the pharmaceutical composition may further include a therapeutic agent selected from a radioisotope, radionuclide, a toxin, a chemotherapeutic agent or a combination thereof.
In one aspect, the application provides methods for treating, preventing or controlling conditions such as cancer, autoimmune diseases, or an infectious disease. In one embodiment, the method includes the steps of administering a pharmaceutical composition comprising a purified multi-specific antibody, the multi-specific antibody-like protein or its fragments, as disclosed herein.
In one aspect, the application provides methods for treating a human subject with a cancer, an autoimmune disease, or an infection. In one embodiment, the method includes the step of administering to the subject an effective amount of the GNC protein such as the purified multi-specific antibody or the multi-specific antibody-like protein or their fragments as disclosed herein.
In one embodiment, the method may further include the step of co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises 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, CD73, or a combination thereof.
In one aspect, the application provides a solution comprising an effective concentration of the GNC protein such as the multi-specific antibody or the multi-specific antibody-like protein or their fragments thereof. In one embodiment, the solution may be 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 present application relates to guidance and navigation control (GNC) proteins and methods of making and using thereof. In some embodiments, the GNC proteins may be multi-specific antibody-like proteins. In some embodiments, the GNC proteins may be multi-specific antibodies, in which cases the GNC proteins may also be referred to as GNC antibodies. In some embodiments, the application provides penta-specific antibody-like proteins and hexa-specific antibody-like proteins. In some embodiments, the application provides penta-specific antibodies and hexa-specific antibodies.
The GNC proteins include the proteins linking multiple functionally independent binding moieties into a single entity that is capable of bringing both effector cells and target cells together (see Applicant's application WO/2019/005642, incorporated herein in its entirety). In one embodiment, these multi-specific binding molecules targeting tumor antigens and immune-activating receptors can utilize similar mechanisms of immune effector cell-mediated killing of tumors at a fraction of the cost. Rather than genetically modifying individual patient T cells, such multi-specific binding molecules can be efficiently manufactured large-scale and administered in a more general off-the-shelf manner. Of the GNC proteins, multi-specific antibodies, such as tetra-specific antibodies, have been shown be able to exert desirable multi-facet GNC effects with structurally and functionally diverse but relatively independent binding domains (see Applicant's application WO/2019/191120, incorporated herein in its entirety).
In one embodiment, the GNC protein may include a multi-specific antibody-like protein comprising a heavy chain and a light chain moiety. The antibody's Fab region is composed of one constant and one variable domain from the heavy and the light chain moiety. The heavy chain may further include three additional antigen-specific binding domains attached to the N-terminal, the C-terminal, or both terminals. The light chain moiety may include one or two additional binding domains attached to the N-terminal, C-terminal, or both terminals.
In some embodiments, the GNC antibodies may be penta-GNC antibodies or hexa-GNC antibodies, as shown in
In one embodiment, the GNC antibodies may include at least one binding domain capable of binding to one surface molecule on a T cell and at least one binding domain capable of binding to one surface antigen on a tumor cell (Table 1). In some embodiments, the surface molecules on a T cell comprise signaling proteins, including without limitation, CD3, NKG2D, and 4-1BB; the surface molecules on a NK cell comprise signaling proteins, including without limitation, NKG2D and 4-1BB; and the surface antigens on a tumor cell comprise tumor antigens, including without limitation, EGFR, HER2, HER3, MSLN, CD19, and PD-L1. In one embodiment, the tumor cells constitute a tumor or a cancer, including without limitation, a solid tumor, a sarcoma, a hematopoietic malignancy, a lung cancer, a pancreatic cancer, a bladder cancer, a cervical cancer, a breast cancer, a leukemia, and a lymphoma.
The GNC antibodies having at least four additional binding domains in addition to the D2 may require structural stability to maintain independent function of binding specificity and affinity of each binding domain. Each additional binding domain may include a (GxSy)n peptide linker, wherein n is an integer from 1 to 10, x is an integer from 1 to 10, and y is an integer from 1 to 10.
In one embodiment, the binding domain such as D1, D2, D3, D4, D5, or D6 may be a single chain variable fragment (scFv), a receptor, or a ligand (Table 1). A scFv domain may be configured to have a fusion of the variable regions of the heavy (VH) and light chain (VL) in either the VH-VL (HL) or VL-VH (LH) orientation. In one embodiment, the scFv domain may be a stapled structure by introducing a disulfide bond between VH44 and VL100 (Kabat). In one embodiment, the VH region for VH3-containing scFv on any light chain moiety has a R19S mutation (Kabat numbering).
The binding domain may be configured to bind to at least one epitope of an antigen, including without limitation, CD3, 4-1BB, EGFR, HER2, HER3, MSLN, CD19, and PD-L1. The amino acid sequences selected to encode the anti-EGFR binding domain may be humanized sequences. In other embodiments, the amino acid sequences selected to encode the anti-CD19 binding domain are humanized sequences.
In one embodiment, the binding domain may be receptors. In one embodiment, the receptor may be NKG2D. In one embodiment, the D2 may include NKG2D.
The binding domain may be ligands for a receptor such as 4-1BBL (a 4-1BB receptor ligand) and 4-1BBL trimer for 4-1BB, a receptor.
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.
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, such as Fab, F(ab′)2, and Fv, so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, chimeric, single chain, multi-specific, multi-effective, human and humanized antibodies. Examples of active antibody fragments that bind to known antigens include Fab, F(ab′)2, scFv, and Fv fragments, as well as the products of a 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 to 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 moiety is comprised of a light chain moiety variable domain (abbreviated as VL) and a light chain moiety 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 heavy and light chain there are binding regions that interacts with the antigen.
The term of “monoclonal” antibody as used herein include “monoclonal mono-specific”, “chimeric”, and “multi-specific” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain moiety 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., PNAS USA, 1984). Monoclonal antibodies can be produced using various methods, including without limitation, mouse hybridoma, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods (see Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al. PLOS One. 2014).
The term “multi-specific” antibody as used herein denotes an antibody that has at least two binding sites each having a binding affinity to an epitope of an antigen. The term “bi-specific, tri-specific, tetra-specific, penta-specific, or hexa-specific” antibody as used herein denotes an antibody that has two, three, four, five, or six antigen-binding sites. For example, the antibodies disclosed herein with five binding sites are penta-specific, with six binding sites are hexa-specific.
The term “guidance and navigation control (GNC)” protein refers to a multi-specific protein capable of binding to at least one effector cell (such as immune cell) antigen and at least one target cell (such as tumor cell, immune cell, or microbial cell) antigen. The GNC protein may adopt an antibody-core structure including a Fab region and Fc region with various binding domains attached to the antibody-core, in which case the GNC protein is also termed GNC antibody. The GNC protein may adopt an antibody-like structure, in which case the Fv fragment may be replaced with a non-antibody based binding domain such as NKG2D, 4-1BBL (a 4-1BB receptor ligand), 4-1BBL trimer for 4-1BB, or a receptor.
The term “GNC antibody” refers to a GNC protein had an antibody structure that is capable of binding to at least one effector cell (such as immune cell) and at least one target cell (such as tumor cell, immune cell, or microbial cell) simultaneously. The term “bi-GNC, tri-GNC, tetra-GNC, penta-GNC, or hexa-GNC” antibody as used herein denotes a GNC antibody that has two, three, four, five, or six antigen-binding sites, of which at least one antigen-binding site has the binding affinity to an immune cell and at least one antigen-binding site has the binding affinity to a tumor cell. In one embodiment, the GNC antibodies disclosed herein have five to six binding sites (or binding domain) and are penta-GNC and hexa-GNC antibodies, respectively. In some embodiments, the GNC antibodies include antibody binding domains (such as Fab and scFv) without the requirement for additional protein engineering in the Fc region. In one embodiment, the GNC antibody may include a Fc region that is engineered to eliminate effector cell function such as ADCC, ADCP, CDC. Mutations include, but are not limited to L234A/L235A/G237A/K322A and L234A/L235A/K322A (Eu numbering). In one embodiment, mutation of the Fc glycosylation site, e.g, N297A (Eu), may be used to prevent glycosylation and disrupt Fc effector functions. In one embodiment, the GNC antibody as used herein comprises symmetric antibodies that do not require Fc engineering to drive proper assembly of the full protein. In contrast, many existing bi-specific and multi-specific antibody formats require a heterodimerizing Fc in order to combine different specificities into asymmetric molecules. In one embodiment, the GNC antibodies additionally have the advantage of retaining bivalency for each targeted antigen. Further in one embodiment, the GNC antibodies have the advantage of avidity effects that result in higher affinity for antigens and slower dissociation rates. This bivalency for each antigen is in contrast to many multi-specific platforms that are monovalent for each targeted antigen, and thus often lose the beneficial avidity effects that make antibody binding so strong.
The term “humanized antibody” 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 Queen et al., Proc. Natl Acad Sci USA, 1989; Hodgson et al., Bio/Technology, 1991). In one embodiment, the “humanized antibody” may be obtained by genetic engineering approach that enables production of affinity-matured humanlike polyclonal antibodies in large animals such as, for example, rabbits (see U.S. Pat. No. 7,129,084).
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.
The term “epitope”, also known as “antigenic determinant”, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, and is the specific piece of the antigen to which an antibody binds.
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 application to moderate or alleviate the disorder to be treated.
The term “tumor antigen” as used herein means an antigenic molecule produced in tumor cells. A tumor antigen may trigger an immune response in the host. In one embodiment, the tumor cells express tumor antigens, including without limitation, tumor-specific antigens (TSA), neoantigens, and tumor-associated antigens (TAA).
The term “specific binding to” or “specifically binds to” or “specific for” a particular antigen or an epitope as used herein means the binding that is measurably different from a non-specific interaction. Specific binding can be measured 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. Specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or an epitope can be exhibited 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, 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 a dissociation rate of a particular antibody-antigen interaction. In some embodiments, a multi-specific antibody that specifically binds to 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 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.
The term “stapled” means two domains are covalently linked. In one embodiment, the two domains may be covalently linked through at least one disulfide bond. For example, a scFv domain that has at least one disulfide bond linking VH and VL is called a stapled scFv; and a Fab region that has at least one disulfide bond linking the light chain moiety and the heavy chain is called a stapled Fab.
While the following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.
In multi-specific GNC antibodies such as tetra-GNC antibodies, the heavy chain may comprise up to three scFv domains plus the Fab region to constitute four binding specificities, whereas the light chain remains unmodified. In penta-GNC antibodies, one scFv domain is added to either N-terminus or C-terminus of the light chain to gain the fifth binding specificity, as shown in
A pair of penta-GNC antibodies (SI-1P1 and SI-1P2) (SEQ ID NO. 1-4 and 5-8, respectively) with identical binding specificities were created for analysing the effect of stapled scFv domains. The heavy chain of the two antibodies comprises αCD3 scFv at D1, αEGFR VH at D2 (in the Fv-CH1-Fc configuration), αPD-L1 at D3, and α4-1BB at D4, and the light chain comprises αEGFR VL and αHER3 scFv at D5 according to the naming system in
Both SI-1P1 and SI-1P2 were cloned into vector pTT5 following a modular cloning strategy using restriction sites HindIII/SalI/NheI/BamHI/BspEI/PacI. These penta-GNC antibody constructs were expressed with acceptable titers using both HEK and ExpiCHO expression systems for 5 and 9 days, respectively, and purified with 5 mL MabSelect protein A columns followed by Size Exclusion using a hiload 16/600 200 pg preparative SEC column on either an Akta Avant or Purifier system. SEC aggregates were analyzed using a waters HPLC linked to multi angle light scattering (MALS, Wyatt systems) to identify correct molecular weight by do/dc calculated methods. Dynamic light scattering (Wyatt systems) was used in the further analysis to determine the melting temperature of the produced protein. With all of the analyses conducted as shown in Table 2, the disulfide bonded, i.e. “stapled”, penta-GNC antibodies displayed more stable characteristics.
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 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 penta-GNC antibody, SI-77P1 (SEQ ID NO. 41-44), harbored R19S mutation in its light chain sequence encoding the anti-CD19 scFv at domain 6, and the hexa-GNC antibodies, SI-55-H11 (SEQ ID NO. 53-56), SI-55H12 (SEQ ID NO. 57-60), SI-77H4 (SEQ ID NO. 61-64), and SI-77H5 (SEQ ID NO. 65-68) harbored R19S mutation in their 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 (4), 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.
Wild-type IgG1 antibodies contain an active Fc domain which binds to Fc gamma receptors on immune cells, as well as C1q, the first component of the complement cascade. These binding capabilities allow antibodies with active Fc to elicit effector functions including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent dependent cytotoxicity toward antigen-bearing cells. However, in the context of T cell redirection, an active Fc domain can exacerbate cytokine release syndrome and cause off-target cytotoxicity (Strohl & Naso, Antibodies, 2019). Therefore, null Fc domains incorporating silencing mutations that weaken binding to Fc gamma receptors and complement can decrease cytokine release syndrome, and even increase efficacy of T cell redirecting antibodies by increasing infiltration into the tumor (Wang et al., Cancer Immunol. Res. 2019). Many point mutations have been introduced to weaken interaction with Fc gamma receptors or C1q, and to lessen Fc effector functions (Saunders, Frontiers Immunol. 2019). Among these, the L234A, L235A, and G237A mutations have been shown to decrease ADCC and ADCP through decreased binding to Fc gamma receptors. The mutation K322A has been shown to decrease binding to C1q, and therefore ablate CDC. Furthermore, mutation of N297A removes the Fc glycosylation site, generating aglycosylated Fc domain that does not interact as strongly with its receptors.
Fc silencing mutations were incorporated into the GNC platform in order to generate therapeutics with mitigated risk of cytokine storm and improved tumor penetration qualities due to less binding of Fc gamma receptors in the periphery. Example molecules contained different null Fc versions to demonstrate that an array of Fc archetypes could be used in the GNC platform. Thus, mutations used to modulate effector function of monoclonal and multi-specific antibodies can also be efficiently incorporated into the GNC platform.
While selecting binding specificities dictates the utility of a multi-specific GNC antibody, optimizing commonly used binding domains may improve the efficacy of the antibody. The penta-GNC and hexa-GNC antibodies (collectively known as GNC antibodies as listed in Table 1) were cloned, expressed, and produced following the similar materials and methods as described for producing SI-1P1 and SI-1P2 antibodies in Example 1.
The anti-CD3 variable domain sequences, 284A10 (Applicant's application No. PCT/US2018/039143, incorporated herein in its entirety), were used as unmodified, stapled (284A10 stapled, SEQ ID NO. 89-92), humanized (284A10 H1, SEQ ID NO. 93-96), or humanized and stapled (284A10 H1 stapled, SEQ ID NO. 97-100) sequences to encode either a scFv (in either VH-VL or VL-VH orientation) or a Fab of the heavy chain monomer. The other anti-CD3 variable domain sequences, 283E3, were identified, cloned, and humanized as 283E3 H1 (SEQ ID NO. 101-104), which were used to encode the Fab region at D2 of the heavy chain monomer for each of seven penta-GNC antibodies as indicated in Table 1.
The anti-PD-L1 variable domain sequences, PL221G5 (Applicant's application No. PCT/US2018/039144, incorporated herein in its entirety), were humanized and used to encode either a “unstapled” or a “stapled” (SEQ ID NO. 105-108) scFv domain at D3 of the heavy chain monomer.
The anti-4-1BB variable domain sequences, 466F6 (Applicant's application No. PCT/US2018/039155 incorporated herein in its entirety), were humanized and used to encode either a “unstapled” or a “stapled” (SEQ ID NO. 109-112) scFv domain at D4 of the heavy chain monomer.
To assess the functionality of the GNC antibodies, the binding affinity of each individual domain of penta-GNC antibodies was carried out by Biolayer Interferometry (Octet 384 system). The penta-GNC antibodies were captures onto the probe using anti-human Fc (AHC tips), and individual epitopes (CD3ε/δ and EGFR were produced, 4-1BB and PD-L1 (Acro Biosystems) were used as analyte to determine the disassociation constant (KD) by kinetic methods (Koff/Kon). As shown in Table 3, all of the binding constants of the individual domains in either SI-1P1 or SI-1P2 were within the reported ranges and the previously determined individual affinities of either monoclonal antibodies or scFv molecules alone.
Octet analysis was used to ensure that GNC antibodies retain their binding to all of their cognate antigens. GNC antibodies were loaded onto AHC sensors for 180 seconds at 10 ug/ml, followed by a 60-second baseline step, a 180-second association step with 100 nM of commercially purchased human antigen, and a 360-second dissociation step. Samples for all steps were in Octet buffer (PBS containing 0.1% Tween 20 and 1% BSA). Fits were performed using a 1:1 binding model to extract affinity KD values, reported in Table 4. The data implies that each binding domain retains its binding affinity when placed at different positions of the GNC antibodies.
To validate the functionality of the GNC antibodies, penta-GNC antibodies were assessed for T cell activation. The T cell activation assay was performed to compare the potency of SI-1P1, which binds to both EGFR and HER3, with that of an EGFR-tetra-GNC antibody (which binds to EGFR but not HER3), a FITC-tetra-specific antibody (which does not bind to either EGFR or HER3), and a bispecific antibody (which only binds to EGFR and HER3). Human pancreatic cancer cells (BxPC3) were used as target cells, which express high levels of EGFR and low levels of HER3 (Table 5). BxPC3 cells were plated in quadruplicate using a BioTek EL406 in 384 well plates at a density of 1500 cells/well after lifting with disassociation reagent (TrypLE Express) and allowed to adhere for 24 hours. Following this, Jurkat CD3 NFAT Effector cells were added at a cellular ratio of 5:1 (Jurkat Lucia Cells, Invivogen) and the GNC antibody was added in a 10-point 10-fold serial dilution from 50 nM to 0.5 fM and incubated for 4 Hours. Readout was performed by the addition of Promega Bright-Glo reagent and luminescence was measured on a Clariostar Plus microplate reader (BMG-Labtech). Data was plotted in log scale with Graphpad prism and fit to a nonlinear variable slope equation (
TDCC is a standard feature of antibody therapy for treating cancer other diseases. To assess the TDCC mediated by the GNC antibodies, SI-1P1 (a penta-GNC antibody capable of binding to tumor antigens EGFR and HER3) was used to compare with control antibodies, including a EGFR-tetra-GNC antibody that only binds to EGFR, a FITC-tetra-specific antibody that does not bind either EGFR or HER3, and a control bispecific antibody that only binds to tumor antigens EGFR and HER3 (Table 6). Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of antibodies were added to a white 384-well plate containing luciferized MDA-MB-231 or HeLa cells (both have high EGFR and low HER3, see Table 5 and plated 24 hours prior and grown at 37° C.) and activated T cells (plated immediately before drug; effector:target=5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values (
An increase number of binding specificities allows the GNC antibodies to bind not only T cells but also subsets of T cells, natural killer cells, and other types of immune cells. On the hand, an added binding specificity may replace the cellular response to or recognition of targeted cells. For example, 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 viral infection or genomic stress such as in cancer. In humans, NKG2D is expressed by NK cells, γδ T cells, and CD8+ αβ T cells. In NK cells, NKG2D serves as an activating receptor, which itself is able to trigger cytotoxicity, whereas on CD8+ T cells the function of NKG2D is to send co-stimulatory signals to activate them. The addition of NKG2D as a binding specificity for the GNC antibodies may improve the cytotoxicity and efficacy of the antibody as a single multi-functional therapeutic agent. In this context, penta-GNC antibodies, SI-49P1 and SI-49P3 (SEQ ID NO. 17-20 and SEQ ID NO. 21-24, respectively), were created by adding NKG2D receptor as a binding domain at D5 (Table 1). The affinity of NKG2D of SI-49P3 (Table 4) for human MICA was founded to be within the expected range, indicating that NKG2D can act as a receptor for the penta-GNC antibody to bind its ligand.
Both SI-49P3 and SI-49P1 are capable of recognizing one tumor antigen via the Fab region while extending multiple binding specificities to CD3, PD-L1, 4-1BB, and NKG2D. To demonstrate that SI-49P3 retains its ability in T cell activation, BxPC3 target cells were plated in quadruplicate using a BioTek EL406 in 384 well plates at a density of 1500 cells/well after lifting with disassociation reagent (TrypLE Express) and allowed to adhere for 24 hours. Following this, Jurkat CD3 NFAT Effector cells were added at a cellular ratio of 5:1 (Jurkat Lucia Cells, Invivogen) and GNC reagent was added in a 10-point 10-fold serial dilution from 50 nM to 0.5 fM and incubated for 4 Hours. Readout was performed by the addition of Promega Bright-Glo reagent and luminescence was measured on a Clariostar Plus microplate reader (BMG-Labtech). Data was plotted in log scale with Graphpad prism and fit to a nonlinear variable slope equation as shown in
To assess TDCC of the NKG2D class of penta-GNC antibodies, SI-49P1 was used. The control antibodies included a tri-GNC antibody lacking the binding specificities to both PD-L1 and 4-1BB, and a tetra-GNC antibody lacking NKG2D receptor. Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of GNC protein were added to a white 384-well plate containing luciferized MDA-MB-231 cells (MICA and mesothelin expression; plated 24 hours prior and grown at 37° C.) and activated T cells (plated immediately before drug; effector:target=5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values of 0.1865 pM (SI-49P1), 0.3433 pM (tri-GNC control antibody), and 5.356 pM (tetra-GNC control antibody) (
To test the utility of antibody-like GNC proteins with natural receptors at position D2, the sequence for human NKG2D (residues F78 through V216) was cloned in place of VH and VL domains at position D2 in the context of expression plasmids encoding SI-49P10 (αCD3×NKG2D×αPD-L1×α4-1BB×αEGFR, SEQ ID NO. 117-200). SI-49P10 was expressed following the materials and methods above, and had exceptionally low aggregation (95.64% peak of interest by analytical SEC) after protein A purification, indicating that the antibody-like GNC proteins containing a non-antibody binding moiety, such as NKG2D receptor, in the D2 position of the heavy chain have the potential to be highly stable. Penta GNC proteins SI-49P6 (αCD3×NKG2D×αPD-L1×α4-1BB×αCD19, SEQ ID 123-126) and SI-49P7 (αCD3×NKG2D×αPD-L1×41BBL trimer×αCD19, SEQ ID 127-130) were similarly cloned, expressed, and purified.
To ensure that the NKG2D dimer retained full functionality, Octet binding to human MICA was assessed. SI-49P10 was loaded onto AHC tips and bound to His-tagged MICA. The extracted KD values confirmed that NKG2D retains binding activity when present in the D2 position (Table 4). SI-49P10 had a KD value of 1.84 nM. As a comparison, SI-49P3 (NKG2D dimer in D5) had a similar KD value of 1.39 nM. The other domains of SI-49P10 also retained high binding affinity to their cognate antigens (Table 4). Similarly, binding of SI-49P6 and SI-49P7 for MICA was determined by loading biotinylated human MICA onto SA tips and observing binding to serial dilution of GNC proteins (0 to 100 nM) as analytes. The KD resulting KD values were 7.763 nM (SI-49P6) and 10.67 nM (SI-49P7), again confirming the retention of target binding by receptor proteins in the D2 position (Table 4). These KD values with antigen as the loaded ligand were slightly lower affinity compared to the experiment in which GNC protein was loaded, possibly due to inactive conformation or incompletely exposed epitope of the MICA protein when it is loaded as ligand. Nevertheless, the potent femtomolar (<1 pM) TDCC elicited by these proteins with NKG2D in D2 position toward MICA-bearing MDA-MB-231 cells (
4-1BB is a co-stimulatory immune checkpoint TNFR receptor expressed by activated T cells and NK cells. Its activation by 4-1BB ligand or by an agonist antibody on CD8+ T cells results in increased proliferation, cytokine production, and survival. To optimize the 4-1BB mediated immune response, 4-1BB activation reporter bioassay was performed to assess the functionality of different domains. The 4-1BB activation assay is based on the methods followed by Promega 4-1BB Bioassay kit (SKU: JA2351). The assay consists of a genetically engineered Jurkat T cell line that expresses human 4-1BB and a luciferase reporter driven by a response element that can respond to 4-1BB ligand/agonist antibody stimulation, called 4-1BB Effector Cells. 4-1BB effector cells are cultured in RPMI-1640 with 10% FBS. Before the assay, the cells are counted and re-plated into 384 well (Corning 3570) at 500 cell/well. 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 4-1BB assay plate was incubated for 6 hours. Readout of the 4-1BB activation curve was accomplished by the use of the Promega Bright-Glo luciferase assay kit. Briefly, 20 uL were added to the 4-1BB assay plate and incubated for ˜15 min before measuring the resultant luminescence on a BMG Clariostar plate reader. Activation curves were analyzed and plotted in GraphPad Software by 4PL curve (
Biological activity of the GNC proteins with NKG2D in D2 position was determined using TDCC assay with MICA-bearing MDA-MB-231 target cells (
Cetuximab is a chimeric mouse/human monoclonal antibody for treating EGFR-expressing metastatic colorectal cancer, non-small cell lung cancer, and head and neck cancer. Humanized antibody is obtained. In this example, humanized sequences encoding anti-EGFR binding (H1, H4, H7, and H7-stapled) (SEQ ID NO. 69-72; 73-76; 77-80, and 81-84, respectively), were cloned into an expression cassette for producing anti-EGFR (D2) penta-GNC antibody (51-77P1) and anti-EGFR (D1) penta-GNC antibodies (SI-55P3, SI-55P4, SI-79P2, SI-79P3, and SI-55P9)(SEQ ID NO. 25-28; 29-32; 45-48; 49-52; 33-36, respectively) as listed in Table 7. Each expression cassette was transfected into 25 mL of ExpiCHO and expressed for 8 days followed by protein-A affinity chromatography for harvesting and purifying each penta-GNC antibody. The antibodies were produced with good titer (Table 7). Analytical SEC data after protein-A purification demonstrates that the penta-GNC antibody containing a humanized anti-EGFR domain can be expressed with low aggregation (Table 7). Octet was used to verify that the penta-GNC antibodies containing humanized anti-EGFR domains, H1, H4, or H7, can bind to human EGFR, respectively (
To produce hexa-GNC antibodies, the humanized anti-EGFR domain, H7 (SEQ ID NO. 77-80), was cloned into an expression cassette for producing anti-EGFR hexa-GNC antibodies. The humanized binding domain was placed at either Fab or as scFv at D1 in hexa-GNC antibodies, SI-77H4 (SEQ ID NO. 61-64) and SI-55H11 (SEQ ID NO. 53-56), respectively. The control antibody, SI-77H5 (SEQ ID NO. 65-68), comprises the anti-EGFR binding Fab region encoded by the Cetuximab mouse sequences. The expression cassette was transfected into 25 mL of ExpiCHO and expressed for 8 days followed by protein A affinity chromatography for harvesting and purifying each hexa-GNC antibody. The hexa-GNC antibodies were produced with good titer (Table 8). Analytical SEC data after protein A purification demonstrates that each hexa-GNC antibody containing a humanized anti-EGFR domain can be expressed with low aggregation (Table 8). Octet was used to verify that each of these hexa-GNC antibodies containing a humanized anti-EGFR domain can bind to human EGFR (Table 4 and 8). The hexa-GNC antibodies were loaded via AHC sensors at 10 μg/ml and bound to a serial dilution (highest 200 nM, 1:2.5 dilutions) or a single 100-nM concentration of His-tagged human EGFR. The resulting global fit to a 1:1 binding model demonstrated that the affinity of each hexa-GNC antibody binding to EGFR was in the low nanomolar range (Table 8).
CD19 is a biomarker for B lymphocyte development and lymphoma diagnosis. CD19-targeted therapies based on T cells that express CD19-specific chimeric antigen receptors (CAR-T) have been utilized for their antitumor abilities in patients with CD19+ lymphoma and leukemia, such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphocytic leukemia. In this context, a humanized CD19 binding domain is desirable.
All computational steps were performed in the Discovery Studio package (Dassault Systemes). First a structural model was generated using the mouse BU12 sequence. Antibody framework regions in the input sequence were identified and aligned to a database of antibody variable domains using Hidden Markov Models (HMM), and this alignment was used to build and score models using the MODELLER software. CDR loop modelling was performed by a structural mapping of the CDRL1 CDRL2 CDRL3 CDRH1 and CDRH2 regions to known canonical classes and loop models were built similarly to the framework. The framework regions from the mouse BU12 antibody were aligned and matched to the closest human germline sequence and CDRs regions were copied into the human sequence with the exception of important structural residues (Vernier residues [Almagro and Fransson, 2008]). Mutations predicted to stabilize the previously build structural model were evaluated computationally by 1000 steps of Steepest Descent with a RMS gradient tolerance of 3, followed by Conjugate Gradient minimization and stabilizing mutations matching frequent human residues were chosen on the basis of individual and combined −ΔΔG versus the initial model. The resulting final sequences were tested for humanness using the Abysis webserver based on the method of Abhinandan and Martin (2007).
To equip penta- or hexa-GNC antibodies with the binding specificity to CD19 for targeting B cell malignancies, the sequences encoding anti-CD19 VL and VH domains were selected from SEQ ID NO. 87, 88, 121, 122, 131, 132 carrying modified VL; and SEQ ID NO. 85, 86, 87, 88 also carrying modified VL along with VH containing R19S mutation (Table 1, also see Example 1 for R19S mutation) and connected with a (G4S)x4 linker to form the anti-CD19 scFv domain. The corresponding gene sequence was cloned into different positions of penta- or hexa-GNC antibodies using restriction digest into the pTT5 expression plasmid for the appropriate heavy or light chain moiety. The anti-CD19 penta-GNC and hexa-GNA antibodies as listed in Table 1 were produced and characterized as described above. Octet analysis of CD19 binding affinity indicated that each GNC antibody retains CD19 binding affinity in an expected range. when placed on the light chain moiety monomer of the GNC antibodies (Table 4).
With the optimized specific binding for EGFR, CD19, and 4-1BB receptor, the penta-GNC antibodies were assessed in TDCC assay. SI-55P9 and SI-55P10 (SEQ ID NO. 33-36 and 37-40, respectively) are a pair of penta-GNC antibodies with identical binding specificities, except SI-55P9 has a humanized anti-EGFR binding domain and SI-55P10 uses 4-1BBL trimer, as to anti-4-1BB binding domain in SI-55P9, to activate 4-1BB signaling. To assess the effect of these differences in TDCC assay, serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of Penta GNC protein SI-1P1, SI-55P9, and SI-55P10 were added to a white 384-well plate containing luciferized BxPC3 (high EGFR expression) cells (plated 24 hours prior and grown at 37° C.) and activated T cells (plated immediately before drug; effector:target=5:1 for SI-1P1 and 7:1 for SI-55P9 and SI-55P10) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. As shown in
Any antibody-based binding domain may be converted to Fab or scFv format and plugged directly into a GNC antibody. For example, the GNC antibodies are characterized by adding the fifth and/or sixth binding domains to the light chain moiety. If the binding specificities on the heavy chain can be dedicated to frequently used targets, such as CD3, PD-L1, and 4-1BB, the utilities of GNC platform may become flexible in terms of selecting targeted tumor antigens and paring the less flexible heavy chain with a desirable light chain moiety. In this context, three tetra-GNC antibodies were selected (from Applicant's application No. PCT/US2019/024105, incorporated herein in its entirety) and evaluated using the in vitro redirected T cell cytotoxicity (RTCC) assay and in vivo human tumor xenograft models.
SI-35E20 is a tetra-GNC antibody capable of binding to 4-1BB (D1), PD-L1 (D2), ROR1 (D3), and CD3 (D4) (Table 1). The ability of SI-35E20 to induce RTCC was determined using live cell imaging of cultures containing PBMC (single donor) and red fluorescence-labeled tumor cells over a 4-day period. PBMC (50,000 cells/mL) were used against NucRed-transduced A549 lung adenocarcinoma cells at a ratio of 4:1 for PBMC and A549. The assay wells were set up in triplicate with 1 nM of SI-35E20 or no GNC (buffer alone) as negative control, and proliferation of target cells was monitored over time for 94 hours. The data shows that SI-35E20 is capable of suppressing the growth of targeted cancer cells over time (
SI-38E17 is a tetra-GNC antibody capable of binding to CD3 (D1), CD19 (D2), PD-L1 (D3), and 4-1BB (D4) (Table 1). To evaluate the effect of SI-38E17-mediated RTCC on cancer cells, Nalm-6 Nuc-GFP (a human leukemic cell line) was used as target cells and PBMC from one donor was used as effector cells. RTCC assay was conducted at E:T ratio=1. At 100 pM, the SI-38E17-mediated RTCC was traced by IncuCyte to detect the proliferation of target cells for 48 hours. SI-38E17 mediated strong RTCC functional activity against Nalm-6 (
SI-39E18 is a tetra-GNC antibody capable of binding to CD3, EGFRvIII, PD-L1, and 4-1BB. The RTCC assay confirms that SI-39E18 elicits more cell killing than vehicle control as shown in
SI-38E17 was tested in a mouse xenograft model to examine its ability to slow tumor growth in vivo (
SI-39E18 was tested in a mouse xenograft model to examine its ability to slow tumor growth in vivo (
Hexa-GNC antibodies were created to explore multi-functionality as a single antibody therapeutics. SI-55H11 (SEQ ID NO. 53-56) is a hexa-GNC antibody having its binding specificities to CD3 (D1), EGFR (D2), PD-L1 (D3), 4-1BB (D4) on the heavy chain monomer, and HER3 (D5) and CD19 (D6) on its light chain moiety monomer (Table 1). The TDCC assay was used to determine the effect of the presence and absence of targeting PD-L1 and 4-1BB on T cell-mediated killing of tumor cells by comparing with a tri-specific antibody targeting CD3 (D1) on T cells and both EGFR (D2) and HER3 (D5) on tumor cells (Table 4). Serial dilutions (0 to 30 nM; 1 to 5 dilution factor) of Tri or Hexa GNC were added to a white 384-well plate containing luciferized BxPC3 (high EGFR expression) cells (plated 24 hours prior and grown at 37° C.) and activated T cells (plated immediately before drug; effector:target=5:1) in a total volume of 50 ul. After an additional 72 hours, 20 ul of Bright-Glo (Promega) was added to wells, and luminescence corresponding to viability of luciferized tumor cells was determined using a CLARIOstar plate reader. Data were fit to a sigmoidal function to calculate EC50 values and maximum killing (
While the attempt to fix the immune targets on the heavy chain may be carried out as shown by Example 12 and 13, the binding domains on the light chain moiety may be dedicated to tumor-specific antigens (TSA), tumor-associated antigens (TAA), as well as neoantigens. In this context, several hexa-GNC antibodies were created and subjected to TDCC assay. The GNC antibodies were assessed to determine if additional tumor-targeting specificity can increase T cell-mediated killing of tumor cells (
The above specification and examples provide a complete description of the structure and use of exemplary embodiments. Although certain embodiments have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention. For example, while the above examples may include binding domains at certain positions, they are provided by way of comparison only and not by way of limitation. It is specifically contemplated by this application that the configuration of binding domains and their positions on the GNC proteins could be in any combination. As such, the illustrative embodiments of the present invention are not intended to be limited to the particular embodiments disclosed. Rather, they include all modifications and alternatives falling within the scope of the disclosure. Further, where appropriate, aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments.
a284A10, see Applicant's application No. PCT/US2018/039143;
b284A10 H1 (SEQ ID NO. 93-100), humanized anti-CD3 variable domain sequences encoding either a scFv domain or the Fab region, in either a “unstapled” or a “stapled” form (SEQ ID NO. 155-108); and
c283E3 H1 (SEQ ID NO. 101-104), humanized anti-CD3 variable domain sequences encoding the Fab region.
dSI-huBU12 VH (SEQ ID NO. 121, 122) and SI-huBU12 H1 VL (SEQ ID NO. 87, 88), humanized anti-CD19 variable domain sequences carrying R19S mutation.
eSI-huBU12 H1 VH (SEQ ID NO. 85, 86) and SI-huBU12 H1 VL (SEQ ID NO. 87, 88), humanized anti-CD19 variable domain sequences.
fSI-huBU12 VH (SEQ ID NO. 121, 122) and SI-huBU12 VL (SEQ ID NO. 131, 132), humanized anti-CD19 variable domain sequences carrying R19S mutation.
HMW % was measured using preparative SEC; melting temperature was measuring using dynamic light scattering
DITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTV
AMDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCQASQSI
SSHLNWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYS
WGNVDNVFGGGTKVEIKGGGGSGGGGSGRSLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWV
SDPMWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPSSVSASVGDRVTITCQASQNI
RTYLSWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGSGTDFTLTISDLEPGDAATYYCQSTYL
GTDYVGGAFGGGTKVEIK
SRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGS
TGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEA
This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 62/931,307 filed Nov. 6, 2019, U.S. Provisional Application Ser. No. 62/984,731 filed Mar. 3, 2020, and 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/US2020/059230 | 11/5/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62931307 | Nov 2019 | US | |
| 62984731 | Mar 2020 | US | |
| 62991042 | Mar 2020 | US |
