EA047669B1 - ANTIBODIES CONJUGATED WITH FATTY ACID MOLECULES AND THEIR APPLICATIONS - Google Patents

Description

Cross reference to related application

This application claims priority to U.S. Provisional Application No. 62/982,476, filed February 2020. This disclosure is incorporated herein by reference in its entirety.

Field of technology

The present invention provides monoclonal isolated antibodies or antigen-binding fragments thereof, wherein the monoclonal antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH); and a light chain variable region (VL); wherein the monoclonal antibody or antigen-binding fragment thereof is capable of specifically binding to a target antigen; wherein an amino acid residue in VH, VL or within twenty (20) amino acids, preferably within five (5) amino acids, of VH or VL is replaced with an amino acid residue capable of conjugation with a fatty acid (FA); and wherein after conjugation with FA on the replaced amino acid residue, the monoclonal antibody or antigen-binding fragment thereof is still capable of specifically binding to the target antigen; and wherein the monoclonal antibody conjugated to FA or an antigen-binding fragment thereof reduces or eliminates specific binding to a target antigen in the presence of physiological levels of albumin (e.g., 35 to 50 mg/mL). The present invention also relates to multispecific antibodies or antigen-binding fragments thereof, wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA. This invention also provides nucleic acids and expression vectors encoding the antibodies, recombinant cells comprising the vectors, and compositions comprising the antibodies. Also provided are methods for producing antibodies, methods for conjugating antibodies to FA, methods for producing compositions comprising conjugated antibodies, and methods for using conjugated antibodies to treat cancer.

Link to the sequence listing provided in electronic form

This application contains a sequence listing that is submitted electronically via EFS-Web as an ASCII sequence listing with the file name 065799.32WO1 Sequence List, a creation date of February 22, 2021, and a size of 29 kb. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

State of the art

T cell attractors are molecules consisting of two binding domains, one of which binds to a tumor-associated antigen (TAA) expressed on the surface of a cancer cell and the other domain binds to a T cell surface molecule to activate the T cell. Although various T cell attractor domains have been used as the activating component, anti-CD3 binding domains have been widely used as part of T cell attractors. Ahtu-CD3 bispecific antibodies are used as immunotherapeutic T cell attractors to recruit T cells to tumor cells to facilitate cancer killing. One of the major concerns of this immuno-oncology approach is the risk of cytokine release syndrome (CRS). Modulating the activity of these agents in activating T cells at sites distant from the tumor microenvironment may help reduce the risk of CRS and other toxic effects.

Fatty acids (FA) exist in high concentrations in circulating blood. Due to their hydrophobic nature, FA bind to blood albumin molecules, which are in the range of 35-50 mg/mL (Peters, T., 1996. All About Albumin: Biochemistry, Genetics and Medical Applications. San Diego, CA: Academic Press Limited). Seven common FA binding sites have been identified on albumin (Bhattacharya et al., J Mol Biol. 2000. 303:721-32; Petitpas et al., J Mol Biol. 2001.314:955-60). Furthermore, it has been suggested that tumors use albumin as an energy source to support their aggressive growth (Merlot et al., Front Physiol. 2014. 5:299), which is consistent with the high rate of albumin catabolism at tumor sites (HRADeC, Br J Cancer, 1958. 12:290-304, Andersson et al., J Surg Res. 1991. 50:156-62, Schilling et al., Int J Rad Appl Instrum B. 1992. 19:685-95; Stehle at al., Crit Rev Oncol Hematol. 1997, 26:77-100). The high turnover of albumin at tumor sites suggests a decrease in albumin concentration in the tumor microenvironment. Thus, albumin levels around some tumor cells are expected to be lower than those in circulating blood. Furthermore, interstitial albumin concentrations have been reported to be significantly lower in adipose tissue and skeletal muscle compared with serum concentrations (Ellmerer et al., Am. J. Physiol. Endocrinol. Metab. 278:E352–E356 (2000)).

By exploiting the lower levels of albumin in tissues such as adipose tissue, skeletal muscle, and the tumor microenvironment compared to circulating blood, it may be possible to exploit the binding of FA to albumin to modulate the activity of therapies that target tissues or sites with low albumin levels. This can be accomplished by using FA conjugated to the active site of the therapy such that when albumin binds to FA, the activity of the therapy is reduced or eliminated. When this approach is applied to immuno-oncology therapies such as anti-CD3 monoclonal and/or bispecific antibodies, it may reduce the risk of CRS and other toxicities in cancer treatments.

- 1 047669

The essence of the invention

In one general aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (VH); and a light chain variable region (VL); wherein the antibody or antigen-binding fragment thereof binds to a target antigen, preferably a human target antigen; wherein an amino acid residue in the VH, VL or within twenty (20) amino acids, preferably within five (5) amino acids, of the VH or VL is replaced with an amino acid residue that is conjugated to a fatty acid (FA); and wherein after conjugation to FA on the replaced amino acid residue, the antibody or antigen-binding fragment thereof still binds to the target antigen; and wherein the antibody conjugated to FA or antigen-binding fragment thereof reduces or eliminates specific binding to the target antigen in the presence of physiological levels of albumin (e.g., 35 to 50 mg/mL). The substituted amino acid residue may, for example, be a cysteine residue or a lysine residue, or a modified amino acid suitable for chemical conjugation.

In some embodiments, the substituted amino acid is at an amino acid residue corresponding to residue 25, 27, 62, 64, 73, 76, 101, 112 or 113 of SEQ ID NO:1, or an amino acid residue corresponding to residue 26, 27, 52, 53, 56 or 67 of SEQ ID NO:2, preferably the substitution is selected from a substitution corresponding to S25C, Y27C, K62C, K64C, K73C, S76C, D101C, S112C or S113C of SEQ ID NO:1 or a substitution corresponding to S26C, S27C, S52C, K53C, S56C or S67C of SEQ ID NO:2, wherein the residues are numbered according to Kabat. In some embodiments, the substituted amino acid is at residue 64 corresponding to SEQ ID NO:1 or at residue 26 corresponding to SEQ ID NO:2, preferably the substitution is selected from the substitution K64C corresponding to SEQ ID NO:1 or the substitution S26C corresponding to SEQ ID NO:2, wherein the residues are numbered according to Kabat.

In some embodiments, the substituted amino acid is at residue 119 or 120 of SEQ ID NO:9, 10, 11 or 12, or residue 121 or 124 of SEQ ID NO:13 or 14, preferably the substitution is selected from S119C or T120C of SEQ ID NO:9, 10, 11 or 12, or S121C or Q124C of SEQ ID NO:13 or 14, wherein the residues are numbered according to EU numbering. In some embodiments, the substituted amino acid is at residue 120 of SEQ ID NO:9, 10, 11 or 12, preferably the substitution is T120C, wherein the residues are numbered according to EU numbering.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is an anti-immune cell modulator (ICM) antibody or antigen-binding fragment thereof and is capable of specifically binding to an ICM, preferably a human ICM. The ICM can be selected, for example, from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa, and other cell surface immunoregulatory molecules.

In some embodiments, the anti-ICM antibody or antigen-binding fragment thereof is an anti-CD3 antibody or antigen-binding fragment thereof and is capable of specifically binding to CD3, preferably human CD3. The isolated anti-CD3 antibody or antigen-binding fragment thereof may, for example, comprise a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOS: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOS: 33, 34, 35, 36, 37, and 38, respectively.

In some embodiments, the substituted amino acid is at residue 25, 27, 62, 64, 73, 76, 101, 112, or 113 in the VH of the anti-CD3 mAb (SEQ ID NO:1 or SEQ ID NO:27) or 26, 27, 52, 53, 56, or 67 in the VL of the anti-CD3 mAb (SEQ ID NO:2 or SEQ ID NO:28), preferably the substitution is selected from S25C, Y27C, K62C, K64C, K73C, S76C, D101C, S112C, or S113C in the VH (SEQ ID NO:1 or 27) or S26C, S27C, S52C, K53C, S56C, or S67C in the VL (SEQ ID NO:2 or 28), wherein the residues are numbered according to Kabat. In some embodiments, the substituted amino acid is at residue 64 in VH (SEQ ID NO: 1 or 27) or 26 in VL (SEQ ID NO: 2 or 28), preferably the substitution is selected from a K64C substitution in VH (SEQ ID NO: 1 or 27) or a S26C substitution in VL (SEQ ID NO: 2 or 28), wherein the residues are numbered according to Kabat.

The isolated anti-CD3 antibody or antigen-binding fragment thereof may, for example, comprise a VH region with the polypeptide sequence of SEQ ID NO: 1 with the amino acid substitution K64C, and a VL region with the polypeptide sequence of SEQ ID NO: 2; or a VH region with the polypeptide sequence of SEQ ID NO: 27 with the amino acid substitution K64C, and a VL region with the polypeptide sequence of SEQ ID NO: 28; or a VH region with the polypeptide sequence of SEQ ID NO: 1, and a VL region with the polypeptide sequence of SEQ ID NO: 2 with the amino acid substitution S26C; or a VH region with the polypeptide sequence of SEQ ID NO: 27, and a VL region with the polypeptide sequence of SEQ ID NO: 28 with the amino acid substitution S26C; or a heavy chain constant domain 1 (CH1) region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12, with the amino acid substitution T120C, and a light chain constant domain (CL) region with a polypeptide sequence selected from SEQ ID NO:13 or 14; or

- 2 047669 a VH region with a polypeptide sequence of SEQ ID NO: 1, a VL region with a polypeptide sequence of SEQ ID NO: 2, a CH1 region with a polypeptide sequence selected from SEQ ID NO: 9, 10, 11 or 12 with the T120C amino acid substitution, and a CL region with a polypeptide sequence selected from SEQ ID NO: 13 or 14; or a VH region with a polypeptide sequence of SEQ ID NO: 27, a VL region with a polypeptide sequence of SEQ ID NO: 28, a CH1 region with a polypeptide sequence selected from SEQ ID NO: 9, 10, 11 or 12 with the T120C amino acid substitution, and a CL region with a polypeptide sequence selected from SEQ ID NO: 13 or 14.

In some embodiments, an isolated multispecific antibody or antigen-binding fragment thereof is provided, wherein the multispecific antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof of the invention, and wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA. The multispecific antibody or antigen-binding fragment thereof may, for example, be a bispecific antibody or antigen-binding fragment thereof.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) and a second antigen-binding arm (Ab2), wherein Ab1 and/or Ab2 comprise a substituted amino acid that is conjugated to FA.

In some embodiments, Ab1 binds to an immune cell modulator (ICM), preferably a human ICM. The ICM can, for example, be selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa, and other cell surface immunoregulatory molecules. In some embodiments, the ICM is CD3, preferably human CD3.

In some embodiments, Ab2 binds to a tumor-associated antigen (TAA), preferably a human tumor-associated antigen (human TAA). For example, the TAA can be DLL3.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising H1 and L1, and a second antigen-binding arm (Ab2) comprising H2 and L2, wherein:

(a) H1 and H2 each comprise the CH1 region of a human IgG1, IgG2, IgG3, or IgG4 and (b) each of L1 and L2 comprises the CL region of a human kappa light chain or a human lambda light chain;

where each H1L1 and H2L2 contains a charge pair selected from the group consisting of the following amino acid substitutions:

(1) G166D/E in CH1 H1 and S114K/R in CL L1, respectively, and G166K/R in CH1 H2 and S114D/E in CL L2, respectively;

(2) T187D/E in CH1 H1 and D/N170K/R in CL L1, respectively, T187K/R in CH1 H2 and D/N170D/E in CL L2, respectively;

(3) S131D/E in CH1 H1 and P119K/R in CL L1, respectively, S131K/R in CH1 H2 and P119D/E in CL L2, respectively;

(4) A129D/E in CH1 H1 and S121K/R in CL L1, respectively, A129K/R in CH1 H2 and S121D/E in CL L2, respectively;

(5) K/R133D/E in CH1 H1 and K207K/R in CL L1, respectively, K/R133K/R in CH1 H2 and K207D/E in CL L2, respectively;

(6) K/R133D/E in CH1 H1 and I/L117K/R in CL L1, respectively, K/R133K/R in CH1 H2 and I/L117D/E in CL L2, respectively;

(7) K/R133D/E in CH1 H1 and F/V209K/R in CL L1, respectively, K/R133K/R in CH1 H2 and F/V209D/E in CL L2, respectively;

(8) G166D/E in CH1 H2 and S114K/R in CL L2, respectively, and G166K/R in CH1 H1 and S114D/E in CL L1, respectively;

(9) T187D/E in CH1 H2 and D/N170K/R in CL L2, respectively, T187K/R in CH1 H1 and D/N170D/E in CL L1, respectively;

(10) S131D/E in CH1 H2 and P119K/R in CL L2, respectively, S131K/R in CH1 H1 and P119D/E in CL L1, respectively;

(11) A129D/E in CH1 H2 and S121K/R in CL L2, respectively, A129K/R in CH1 H1 and S121D/E in CL L1, respectively;

(12) K/R133D/E in CH1 H2 and K207K/R in CL L2, respectively, K/R133K/R in CH1 H1 and K207D/E in CL L1, respectively;

(13) K/R133D/E in CH1 H2 and I/L117K/R in CL L2, respectively, K/R133K/R in CH1 H1 and I/L117D/E in CL L1, respectively; or (14) K/R133D/E in CH1 H2 and F/V209K/R in CL L2, respectively, K/R133K/R in CH1 H1 and F/V209D/E in CL L1, respectively.

- 3 047669

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:15, a VL region with the polypeptide sequence of SEQ ID NO:17, a CH1 region with the polypeptide sequence of SEQ ID NO:16, and a CL region with the polypeptide sequence of SEQ ID NO:18.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:19, a VL region with the polypeptide sequence of SEQ ID NO:21, a CH1 region with the polypeptide sequence of SEQ ID NO:20, and a CL region with the polypeptide sequence of SEQ ID NO:22.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:29, a VL region with the polypeptide sequence of SEQ ID NO:30, a CH1 region with the polypeptide sequence of SEQ ID NO:16, and a CL region with the polypeptide sequence of SEQ ID NO:18.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:31, a VL region with the polypeptide sequence of SEQ ID NO:32, a CH1 region with the polypeptide sequence of SEQ ID NO:20, and a CL region with the polypeptide sequence of SEQ ID NO:22.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises:

(a) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO: 15, a VL region with a polypeptide sequence of SEQ ID NO: 17, a CH1 region with a polypeptide sequence of SEQ ID NO: 16, and a CL region with a polypeptide sequence of SEQ ID NO: 18; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO: 23, a VL region with a polypeptide sequence of SEQ ID NO: 25, a CH1 region with a polypeptide sequence of SEQ ID NO: 24, and a CL region with a polypeptide sequence of SEQ ID NO: 26;

(b) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:19, a VL region with a polypeptide sequence of SEQ ID NO:21, a CH1 region with a polypeptide sequence of SEQ ID NO:20, and a CL region with a polypeptide sequence of SEQ ID NO:22; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26;

(c) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:29, a VL region with a polypeptide sequence of SEQ ID NO:30, a CH1 region with a polypeptide sequence of SEQ ID NO:16, and a CL region with a polypeptide sequence of SEQ ID NO:18; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26; or (d) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:31, a VL region with a polypeptide sequence of SEQ ID NO:32, a CH1 region with a polypeptide sequence of SEQ ID NO:20, and a CL region with a polypeptide sequence of SEQ ID NO:22; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26.

In some embodiments, the isolated antibody or antigen-binding fragment thereof is conjugated to an FA at a substituted amino acid residue. The FA can be, for example, selected from an FA with 6 carbon atoms, 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms, or 18 carbon atoms, or any number of carbon atoms in between. In some embodiments, the FA is selected from an FA with 14 carbon atoms or 18 carbon atoms, or any number of carbon atoms in between.

In some embodiments, the FA comprises a linker for conjugation with a substituted amino acid residue. The linker, for example, can be selected from a peptide linker or a polyethylene glycol (PEG) linker. The peptide linker, for example, can consist of less than 50 amino acids.

In some embodiments, the FA conjugated to the antibody or antigen-binding fragment thereof is capable of binding albumin, wherein the binding of albumin to the FA results in a partial or complete block of binding between the target antigen and the antibody or antigen-binding fragment thereof. In some embodiments, where the isolated antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, the binding of albumin to the FA on the Ab1 arm does not affect the binding of the Ab2 arm to its antigen, or the binding of albumin to the FA on the Ab2 arm does not affect the binding of the Ab1 arm to its antigen. In some embodiments, the isolated antibody or antigen-binding fragment thereof conjugated to FA has a reduced ability to activate T cells when bound to albumin, compared to the isolated antibody or antigen-binding fragment thereof conjugated to FA that does not bind to albumin.

Also provided are isolated nucleic acids encoding the isolated antibodies or antigen-binding fragments thereof of the invention.

Also provided are vectors containing isolated nucleic acids encoding isolated antibodies or antigen-binding fragments thereof according to the invention.

Also provided are host cells containing the vectors of the invention.

Also provided are pharmaceutical compositions comprising an isolated antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise an isolated antibody or antigen-binding fragment thereof conjugated to FA and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions comprise an isolated antibody or antigen-binding fragment thereof conjugated to FA, wherein FA is bound to albumin, and a pharmaceutically acceptable carrier.

Also provided are methods for treating cancer in a subject in need thereof, wherein the methods comprise administering to the subject the pharmaceutical compositions of the invention. Cancer can be selected, for example, from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other hematological malignancies.

Methods for producing an isolated antibody or antigen-binding fragment thereof according to the invention are also provided. The methods include culturing a cell containing a nucleic acid encoding the antibody or antigen-binding fragment thereof under conditions that ensure the production of the antibody or antigen-binding fragment thereof, and, optionally, isolating the antibody or antigen-binding fragment thereof from the cell or culture.

Also provided are methods for producing an isolated antibody or antigen-binding fragment thereof conjugated to FA according to the invention. The methods comprise conjugating FA to the antibody or antigen-binding fragment thereof at a substituted amino acid residue. Also provided are methods for producing an isolated antibody or antigen-binding fragment thereof conjugated to FA and bound to albumin, wherein the methods comprise contacting the isolated antibody or antigen-binding fragment thereof conjugated to FA with albumin.

Methods for producing a pharmaceutical composition containing an isolated antibody or antigen-binding fragment thereof according to the invention are also provided. The methods include combining the antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to produce the pharmaceutical composition.

Also provided are methods comprising contacting albumin with a conjugate comprising FA covalently linked, optionally via a linker, to an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof in the conjugate is capable of specifically binding to a target antigen, the FA in the conjugate is capable of binding to albumin, and the binding of albumin to FA results in a partial or complete block of binding between the target antigen and the antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more substituted amino acid residues in a VH, VL, or at a distance of twenty (20) amino acids, preferably at a distance of five (5) amino acids, from a VH or VL, and the one or more substituted amino acid residues are covalently linked, optionally via a linker, to FA. In some embodiments, the contacting step comprises administering a pharmaceutical composition comprising a conjugate to a subject in need of treatment of a tumor, wherein the tumor comprises a target antigen. In some embodiments, albumin has a higher metabolism in the tumor microenvironment compared to circulating blood, and/or albumin is present in the tumor microenvironment at a level lower than the level of albumin in the circulating blood of the subject.

Brief description of the drawings

The above summary of the invention, as well as the following detailed description of the preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. However, it should be understood that the application is not limited to the precise embodiments shown in the drawings.

Figures 1A-1E show schematically the structures of a monoclonal antibody (mAb) (Figure 1A) and a bispecific antibody (bsAb) (Figures 1B and 1C) with a fatty acid (FA) molecule conjugated to the VH region, an illustration of the mechanism of action of the FA-conjugated bispecific antibody in vivo (Figure 1D), and a scheme for identifying FA-conjugated antibodies (Figure 1E). Figures 1A-1C are schematically shown for illustrative purposes only, since the conjugation site can be at other suitable sites in the Fab region, including the VL region and CL region. Furthermore, both arms of the bispecific antibody can be conjugated in Figures 1B-1C. FA can potentially modulate the antigen-binding activity of the conjugated arm to varying degrees. In Figures 1A shows that the FA molecule is conjugated to the VH region of both arms; Fig. 1B shows that the FA molecule is conjugated to the VH region of one of the arms of the bispecific antibody; Fig. 1C shows that the FA molecule is conjugated to the CH1 region of one of the arms of the bispecific antibody. The antigen-binding activity of the conjugated arm is expected to be modulated by albumin, since albumin bound to the conjugated FA can completely or partially block the binding of the target antigen by the conjugated arm. Fig. 1D illustrates the mode of action of the FA-conjugated bispecific antibody for use in engaging T cells in target cancer cells in vivo. When both arms of the bispecific antibody are conjugated with FA, the same purpose can be achieved; However, the modulation by albumin in this case can be increased compared to bispecific antibodies with conjugation of only one arm. Furthermore, Fig. 1D shows how the albumin level regulates the antigen-binding activity of the bispecific antibody conjugated to FA. Fig. 1E shows the specific steps of identifying mAb or bsAb conjugated to FA. Albumin-dependent activity refers to the fact that the activity of the conjugated antibody is modulated by albumin (i.e., high concentrations of albumin reduce or completely block the antigen-binding activity).

Figures 2A-2D show the amino acid sequences of exemplary antibodies. Figure 2A shows the amino acid sequence of the VH region of an anti-CD3 antibody (SEQ ID NO:1). Figure 2B shows the amino acid sequence of the VL region of an anti-CD3 antibody (SEQ ID NO:2). Figure 2C shows the amino acid sequences of the CH1 regions of human IgG1 (SEQ ID NO:9), IgG2 (SEQ ID NO:10), IgG3 (SEQ ID NO:11), and IgG4 (SEQ ID NO:12). Figure 2D shows the amino acid sequences of the CL regions of human kappa (SEQ ID NO:13) and lambda (SEQ ID NO:14) light chains. The CDRs determined by a combination of the IMGT and Kabat methods are highlighted in gray. * represents sites of known allelic variations.

Figures 3A-3G show examples of selected amino acid residues for substitution and conjugation to FA in an anti-CD3 monoclonal antibody (mAb). Figure 3A shows a three-dimensional modeling of the Fab region (containing VH, CH1, VL, and CL) in the anti-CD3 mAb to identify potential cysteine knockin sites for FA conjugation. Four sites in the 3-D structure (LC_S26, LC_S31, HC_K64, and HC_T120) are shown as examples (LC: light chain; HC: heavy chain). Additional sites are shown in Table 3. Figures 3B-3G show graphs demonstrating binding of anti-CD3 mAbs with cysteine knockins to CD3 on Jurkat cells. MFI: mean fluorescence intensity. Ahtu-CD3 mAb, wild-type anti-CD3 mAb.

Figures 4A–4C show the structures of FA molecules for conjugation with anti-CD3 mAbs and the mass spectrometry (MS) profiles of FA-conjugated anti-CD3 mAbs. Figure 4A shows the structures of FA molecules for conjugation. All FA molecules were conjugated via a PEG linker. Figure 4B shows the MS profiles of mAbs (LC_S26C, HC_K64C, and HC_T120C) conjugated with C18 FA. Figure 4C shows the MS profiles of HC_K64C mAb conjugated with C6, C10, and C14 FA, respectively. Expected, expected mass after deconvolution; Obs, observed mass after deconvolution. LC_S26C is an anti-CD3 mAb in which the serine at position S26 of the light chain is replaced by cysteine; all other cysteine-added mAbs follow the same naming convention.

Figures 5A-5C show the effect of albumin on the binding of C18-FA-conjugated anti-CD3 mAb to CD3 on Jurkat cells. The assay was performed in the absence or presence of 50 mg/ml bovine serum albumin (BSA). Figure 5A, conjugated LC_S26C; Figure 5B, conjugated HC_K64C; Figure 5C, conjugated HC_T120C.

Figures 6A-6C show the effect of different albumin concentrations on T cell activation by C18-FA-conjugated anti-CD3 mAbs. Figure 6A, conjugated LC_S26C; Figure 6B, conjugated HC_K64C; Figure 6C, conjugated HC_T120C. The assay medium contains 1% FBS; the concentration of BSA indicated represents the BSA added to the assay medium.

Figures 7A–7C show the effect of different albumin concentrations on T cell activation by C6, C10, and C14 FA-conjugated anti-CD3 mAbs, respectively. In Figure 7A, C6 FA-conjugated HC_K64C; in Figure 7B, C10 FA-conjugated HC_K64C; in Figure 7C, C14 FA-conjugated HC_K64C. Assay medium contains 1% FBS; the indicated concentration of BSA represents BSA added to the assay medium; Control, no BSA added.

- 6 047669

Fig. 8A-8C show the purity of purified aHTu-DLL3/aHTu-CD3 bispecific antibody bsAb HC_K64C, where the K64 residue on the HC arm of anti-CD3 is replaced by cysteine. Fig. 8A shows the result of HPLC CHEM analysis of purified bsAb HC_K64C with some impurity standards; Fig. 8B shows the result of HPLC RMSE analysis of purified bsAb HC_K64C with some impurity standards; Fig. 8C shows the result of HPLC SEC analysis of purified bsAb HC_K64C. Ahtu-CD3 convex homodimer/half mol, impurity standard, which is protein A purified from the medium of cells transfected with anti-CD3 HC and anti-CD3 LC; anti-DLL3 hollow homodimer/half mol, impurity standard which is Protein A purified from the medium of cells transfected with anti-DLL3 HC and anti-DLL3 LC; 2x anti-CD3 LC mismatch, impurity standard which is Protein A purified from the medium of cells transfected with anti-CD3 HC, anti-CD3 LC, and anti-DLL3 HC; 2x anti-DLL3 LC mismatch, impurity standard which is Protein A purified from the medium of cells transfected with anti-CD3 HC, anti-DLL3 HC, and anti-DLL3 LC. Half mol, half an IgG molecule with only one HC and one LC.

Figures 9A-9C show the purity of the purified anti-DLL3/anti-CD3 bispecific antibody bsAb HC_T120C, wherein the T120 residue on the HC arm of anti-CD3 is replaced by cysteine. Figure 9A shows the result of HPLC HBV analysis of the purified bsAb HC_T120C with some impurity standards; Figure 9B shows the result of HPLC RMSE analysis of the purified bsAb HC_T120C with some impurity standards; Figure 9C shows the result of HPLC SEC analysis of the purified bsAb HC_T120C. Ahtu-CD3 convex homodimer/half mol, impurity standard, which is protein A purified from the medium of cells transfected with anti-CD3 HC and anti-CD3 LC; anti-DLL3 hollow homodimer/half mol, impurity standard which is Protein A purified from the medium of cells transfected with anti-DLL3 HC and anti-DLL3 LC; 2x anti-CD3 LC mismatch, impurity standard which is Protein A purified from the medium of cells transfected with anti-CD3 HC, anti-CD3 LC, and anti-DLL3 HC; 2x anti-DLL3 LC mismatch, impurity standard which is Protein A purified from the medium of cells transfected with anti-CD3 HC, anti-DLL3 HC, and anti-DLL3 LC. Half mol, half an IgG molecule with only one HC and one LC.

Fig. 10A-10B show the purity of the purified anti-DLL3/anti-CD3 bispecific antibodies conjugated to fatty acid molecules. Fig. 10A shows the result of the HPLC-CHG analysis of the purified anti-DLL3/anti-CD3 bispecific antibodies conjugated to fatty acid molecules; Fig. 10B shows the result of the HPLC-SEC analyses of the purified anti-DLL3/anti-CD3 bispecific antibodies conjugated to fatty acid molecules. bsAb HC_T120C_C18 refers to the anti-DLL3/anti-CD3 bispecific antibody bsAb HC_T120C conjugated to C18 FA; other conjugated bispecific antibodies follow the same naming convention.

Figure 11 shows the result of cross-linking analysis of SHP-77 and Jurkat cells with unconjugated and conjugated anti-DLL3/anti-CD3 bispecific antibodies in the presence or absence of blocking antibodies. The blocking anti-DLL3 mAb is a mAb version of the anti-DLL3 arm; the blocking anti-CD3 mAb is a mAb version of the anti-CD3 arm (without knocked-in cysteine). bsAb WT, wild-type anti-DLL3/anti-CD3 bispecific antibody (without knocked-in cysteine); SHP-77+Jurkat control, the assay was performed with cells without added antibodies.

Figures 12A, 12B show the results of T cell receptor-CD3 (TCR/CD3) complex activation on Jurkat cells in the presence of SHP-77 cells (expressing DLL3) mediated by unconjugated and conjugated anti-DLL3/anti-CD3 bispecific antibodies. Blocking anti-DLL3 mAb was used to inhibit activation to demonstrate that Jurkat cell activation requires simultaneous binding of SHP-77 cells by the bispecific antibodies. The assay medium contains 0.5% FBS.

Figures 13A-13B show the effects of BSA on TCR/CD3 complex activation on Jurkat cells in the presence of SHP-77 cells (expressing DLL3; also known as the target cell) mediated by unconjugated and conjugated anti-DLL3/anti-CD3 bispecific antibodies. The assay medium contains 0.5% FBS; the concentration of BSA indicated represents BSA added to the assay medium.

Fig. 14 shows the result of the ELISA assay used to evaluate the effect of BSA on the antigen-binding activity of the anti-DLL3 conjugated bispecific antibody arm. Ahtu-DLL3 F(ab') ₂ was used as a control for inhibition of DLL3 binding by the anti-DLL3 conjugated bispecific antibody arm.

Detailed description of the invention

Various publications, articles, and patents are cited or described in the prior art and throughout the specification; each of these references is incorporated herein by reference in its entirety. Discussion of documents, acts, materials, devices, articles, or the like that have been included in this specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions described or claimed.

- 7 047669

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, some terms used herein have the meanings specified in the description.

Unless otherwise specified, any numerical values such as a concentration or concentration range described herein are to be understood as modified in all instances by the term "about". Thus, a numerical value typically includes ±10% of the stated value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Similarly, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the term "numeric range" expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges, and fractions of values, unless the context clearly indicates otherwise.

Unless otherwise indicated, the term at least preceding a series of elements should be understood to refer to each element in the series. One skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be included in the present invention.

As used herein, the terms "comprises," "includes," "including," "has," "having," "contains," or "containing," or any variation thereof, shall be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, "or" refers to an inclusive or rather than an exclusive or. For example, a condition A or B is satisfied by any of the following conditions: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

The connecting term and/or as used herein between several specified elements is understood to cover both separate and combined variants. For example, when two elements are combined and/or, the first variant refers to the applicability of the first element without the second. The second variant refers to the applicability of the second element without the first. The third variant refers to the combined application of the first and second elements. Any of these variants is understood to fall within this meaning and therefore satisfy the requirement of the term and/or as used herein. The simultaneous application of more than one of the variants is also understood to fall within this meaning and therefore satisfy the requirement of the term and/or.

As used herein, the term "consists of" or variations such as "consists of" or "consisting of" as used in the description and claims indicate the inclusion of any specified integer or group of integers, but that no additional integer or group of integers may be added to the specified method, structure or composition.

As used herein, the term "consists essentially of" or variations such as "consists essentially of" or "consisting essentially of" as used in the specification and claims indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not substantially alter the basic or novel properties of the recited method, structure, or composition. See M.P.E.P. § 2111.03.

As used herein, the term subject means any animal, preferably a mammal, most preferably a human. The term mammal as used herein includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

The words right, left, bottom and top refer to the directions in the drawings to which reference is made.

It should also be understood that the terms about, approximately, generally, essentially and similar terms, when used herein with respect to a size or characteristic of a component of the preferred invention, indicate that the described size/characteristic is not a strict limit or parameter and does not exclude minor variations therefrom that are the same or similar in functionality, as will be understood by those skilled in the art. At a minimum, such references that include a numerical parameter should include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), will not change the last significant digit.

As used in this document, the terms different heavy chains or different light chains,

- 8 047669 used in the description and claims indicate that the heavy chains or light chains have sequences that are not identical to each other.

The terms identical or proportion of identity in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-DL3 antibodies, anti-CD3 antibodies, anti-CD3/anti-DL3 bispecific antibodies, DLL3 polypeptides and the polynucleotides that encode them, and CD3 polypeptides and polynucleotides that encode them) refer to two or more sequences or subsequences that are the same or have a specified proportion of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, when measured using one of the following sequence comparison algorithms, or by visual inspection.

To compare sequences, typically one sequence acts as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, subsequence coordinates are assigned if necessary, and the sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the proportion of sequence identity for the test sequence(s) relative to the reference sequence based on the assigned program parameters.

Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), using computerized versions of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see, in general, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms suitable for determining the proportion of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403–410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389–3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when matched with a word of the same length in a database sequence. T is referred to as the neighbor word score threshold (Altschul et al., supra). These initial neighbor word matches act as seeds to initiate a search to find longer HSPs containing them. The word matches are then expanded in both directions along each sequence as much as the combined alignment score can be increased.

The cumulative score is calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Expansion of word hits in each direction stops when: the cumulative alignment score falls by X from the maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating the proportion of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An additional indication that two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid

- 9 047669 is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

As used herein, the term polynucleotide, synonymously referred to as a nucleic acid molecule, nucleotides, or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide refers to triple-stranded regions comprising RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. Modified bases include, for example, tritylated bases and unusual bases such as inosine. DNA and RNA may be modified in a variety of ways; thus, polynucleotide encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides that commonly occur in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. Polynucleotide also encompasses relatively short chains of nucleic acids, often called oligonucleotides.

As used herein, the term vector is a replicon into which another nucleic acid segment can be functionally inserted to cause replication or expression of the segment.

As used herein, the term "host cell" refers to a cell comprising a nucleic acid molecule of the invention. The host cell may be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, the host cell is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, the host cell is a progeny or potential progeny of such a transfected cell. The progeny of the cell may or may not be identical to the parent cell, such as due to mutations or environmental influences that may occur in subsequent generations, or due to the integration of the nucleic acid molecule into the host cell's genome.

As used herein, the term expression refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses the translation of RNA into one or more polypeptides and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed monoclonal or bispecific antibody may be in the cytoplasm of the host cell, in an extracellular environment such as cell culture growth medium, or may be anchored to the cell membrane.

As used herein, the terms peptide, polypeptide, or protein may refer to a molecule composed of amino acids and may be recognized by those skilled in the art as a protein. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms peptide, polypeptide, and protein may be used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass a polymer of an amino acid that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, amino acids of non-natural origin, etc.), as well as other modifications known in the art.

The peptide sequences described herein are written according to the usual convention that the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, the L-form of the amino acid is shown unless otherwise indicated.

As described herein, the term CD3 refers to cluster of differentiation 3, which is a multi-subunit protein complex that functions as a coreceptor for the T cell receptor (TCR) (Dong et al., Nature 573(7775): 546–552 (2019)). Binding of the TCR to peptide-MHC (pMHC) on the surface of target cells induces clustering of the TCR-10 047669

CD3 and activates intracellular signaling mediated by the CD3 ζ chain (Annu Rev Immunol. 27:591–619 (2009)). CD3 is required for T cell activation, and its pMHC-independent activation by therapeutics such as CAR T cells and CD3-based T cell activators is highly effective in mobilizing T cells to kill tumor cells (Brown and Mackall, Nat Rev Immunol 19(2):73–74 (2019) and Clynes and Desjarlais, Annu Rev Med 70:437–450 (2019)). The representative amino acid sequence of the human CD3 epsilon subunit has been submitted to GenBank under accession number NP_000724.1.

As described herein, the term DLL3 refers to delta-like canonical Notch ligand 3 (DLL3), also known as delta-like 3 or delta-like protein 3, which is required for somite segmentation during early development (Dunwoodie et al., Development 129:1795-806 (2002)). Unlike the mammalian Notch family ligands DLL1, DLL4, JAG1, and JAG2, which all activate Notch receptor signaling in trans (Ntziachristos et al., Cancer Cell 25(3):318–34 (2014)), DLL3 is predominantly localized to the Golgi apparatus and fails to activate Notch signaling (Chapman et al., Hum Mol Genet 20(5):905–16 (2011) and Geffers et al., J Cell Biol 178(3):465–76 (2007)). During normal development, DLL3 inhibits activation of both cis- and trans-acting Notch pathways by interacting with Notch and DLL1 (Chapman et al., Hum Mol Genet 20(5):905–16 (2011)). DLL3 is typically absent or present at very low levels in normal adult human tissues, with the exception of the brain, but is overexpressed in lung cancer, testicular cancer, glioma, and melanoma samples (Uhlen et al., Science 357(6352): eaan2507 (2017)). Additionally, DLL3 is detected on the surface of small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) tumor cells (Saunders et al., Sci Transl Med 7(302):302ra136 (2015) and Sharma et al., Cancer Res 77(14):3931–41 (2017)), making it a potential target for monoclonal antibodies for anticancer therapy. Therefore, a monoclonal anti-DLL3 antibody can be used to specifically target tumor cells expressing DLL3 and be used as a potential anticancer therapeutic agent. The term human DLL3 refers to DLL3 derived from humans. The typical amino acid sequence of human DLL3 is submitted to GenBank under accession number NP_058637.1.

Tumor-associated antigen (TAA), as described herein, refers to any peptide and/or cell surface antigen, or a combination of a peptide and/or cell surface antigen and a post-translational modifying moiety (such as glycosylation), that is present in tumors at a higher level than in normal tissues. Certain tumor-associated antigens that are present specifically in tumors are also known as tumor-associated antigens (TSA). Examples of tumor-associated antigens include viral proteins encoded by oncogenic viruses; mutated oncoproteins or tumor suppressors; normal proteins overexpressed on and/or in tumor cells; post-translational modifications of cell surface proteins; oncofetal proteins whose expression is normally restricted during developmental stages but not in adult tissues; and cell type-specific proteins whose expression is restricted to non-essential tissues.

A fatty acid, as described herein, refers to a chemical molecule consisting of hydrocarbon chains terminating in carboxylic acid groups, typically with 6 to 22 carbon atoms. For the purposes of the present invention, various derivatives of fatty acids are also considered fatty acids due to their ability to bind to albumin. Fatty acids and their derivatives are the main components of lipids and impart hydrophobic properties to them. The length and degree of saturation of the hydrocarbon chain vary among fatty acids, which determines the physical properties associated with them. Types of fatty acids include unsaturated fatty acids (polyunsaturated and monounsaturated) and saturated fatty acids; saturated fatty acids are saturated with hydrogen and are generally straight hydrocarbon chains with an even number of carbon atoms.

An immune cell modulator (ICM), as described herein, refers to any cell surface molecule, such as a protein, that is expressed on the surface of immune cells and regulates immune cell function. ICMs include stimulatory molecules and inhibitory molecules. A stimulatory ICM can mediate immune cell activation when a specific antibody or antigen-binding moiety with certain characteristics specifically binds to a stimulatory ICM. An inhibitory ICM suppresses immune cell activity when bound to a ligand/interacting partner, which can be blocked by a specific antibody or antigen-binding moiety with certain characteristics that lead to immune cell activation. These immune cells can be T cells, NK cells, macrophages, or other types of immune system cells. Examples of ICMs include, but are not limited to, CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules.

- 11 047669

As used herein, the term "complete block" or "complete block" refers to complete inhibition of binding of a target antigen (e.g., an ICM such as CD3) to a target antigen-binding domain (e.g., a monoclonal or bispecific antibody or antigen-binding fragment thereof). Complete inhibition of target antigen binding means no binding (e.g., 0% binding) of the target antigen to the target antigen-binding domain.

As used herein, the term "partial block" or "partial block" refers to an incomplete inhibition of binding of a target antigen (e.g., an ICM such as CD3) to a target antigen-binding domain (e.g., a monoclonal or bispecific antibody or antigen-binding fragment thereof). Incomplete inhibition of target antigen binding means that there is at least some binding (e.g., 1% to 99% binding) of the target antigen to the target antigen-binding domain.

As used herein, the term specific binding refers to significant binding of a target antigen to an antibody or antigen-binding fragment thereof compared to a control antigen, and/or significant binding of a target antigen to an antibody or antigen-binding fragment thereof compared to a control antibody or antigen-binding fragment, wherein the control antigen differs from the target antigen by sequence and/or structure comparison, and the control antibody or antigen-binding fragment significantly and selectively binds only to its corresponding antigen that differs from the target antigen by sequence and/or structure comparison.

Antibodies.

The invention generally relates to monoclonal antibodies (mAbs) (e.g., anti-ICM mAbs, such as anti-CD3 mAbs) or bispecific antibodies (bsAbs) (e.g., anti-CD3/anti-DLL3 bsAbs) comprising a fatty acid (FA) molecule conjugated to or adjacent to an antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) (e.g., at the VH, VL, or within twenty (20) amino acids, preferably within five (5) amino acids, of either the VH or VL). The conjugation site is a reactive residue at or adjacent to the antigen-binding domain and may be knocked in with a cysteine or other reactive amino acid. The location of the conjugation site is identified such that the cysteine (or other reactive amino acid) knockin or conjugated FA does not abolish the target antigen (e.g., ICM such as CD3) binding activity of the antigen binding domain. The conjugated FA can bind to an albumin molecule, and the bound albumin molecule can occupy a significant space between the antigen binding domain and the target antigen (e.g., ICM such as CD3). The bound albumin molecule can spatially interfere with binding of the target antigen to the antigen binding domain, resulting in a decrease or complete block in binding of the target antigen to the antigen binding domain. An FA-conjugated mAb or an FA-conjugated arm of a bsAb can be directed against an immune cell modulator (ICM), which upon binding to the antibody can lead to activation of immune cells. Examples of ICMs include, but are not limited to, CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa, and other cell surface immunoregulatory molecules. Thus, the immune cell activating activity of conjugated mAbs or bsAbs can be regulated by albumin bound to the conjugated FA. The degree of regulation depends on the concentration of albumin around the conjugated mAbs or bsAbs, the length of the FA molecule, and the specific location of the conjugation site. The invention also relates to multispecific antibodies or antigen-binding fragments thereof, wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA.

Conjugated FA on a mAb or bsAb or multispecific antibody can bind to circulating albumin in the blood, which may serve to reduce or block binding of the conjugated mAb or bsAb to the target antigen on T cells (e.g., ICM such as CD3), resulting in partial or complete inhibition of T cell activation. In the tumor microenvironment, where the turnover rate of albumin is higher than in circulating blood and local albumin levels are expected to be lower than in circulating blood, conjugated antibodies have less or no albumin bound to them, which may result in increased binding of the target antigen (e.g., CD3) and T cell activation. Additionally or alternatively, higher albumin turnover in the tumor microenvironment may reduce the levels of albumin-bound mAbs or bsAbs or multispecific antibodies and expose the antigen-binding domain, resulting in increased target antigen (e.g., CD3) binding and T cell activation. Conjugated antibodies may have in vivo safety advantages and may be used for therapeutic purposes. Conjugated bsAbs may also be used as T cell attractors or other immune cell attractors, where one arm contains

- 12 047669 antigen-binding domain against tumor-associated antigen (TAA), and the other arm contains a conjugated antigen-binding region of anti-ICM (e.g. anti-CD3 antigen-binding region).

The term antibody as used herein is used in a broad sense and includes immunoglobulins or antibody molecules, including human, humanized, composite and chimeric antibodies and antibody fragments, which are monoclonal or polyclonal. Antibodies are typically proteins or peptide chains that have binding specificity for a particular antigen. The structures of antibodies are well known. Immunoglobulins can be classified into five major classes (i.e., IgA, IgD, IgE, IgG and IgM) based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further divided into IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4 isotypes. Accordingly, the antibodies of the invention can belong to any of the five major classes or their corresponding subclasses. Preferably, the antibodies of the invention are IgG1, IgG2, IgG3 or IgG4. Light chains of vertebrate species antibodies can be classified into one of two distinct types, namely, kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, antibodies of the invention can comprise a kappa or lambda light chain constant domain. According to particular embodiments, antibodies of the invention comprise heavy and/or light chain constant regions of rat or human antibodies. In addition to the heavy and light chain constant domains, the antibodies comprise an antigen-binding region consisting of a light chain variable region and a heavy chain variable region, each of which comprises three domains (i.e., complementarity determining regions 1-3; CDR1, CDR2 and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2 and HCDR3.

Several systems are used to number amino acid residues in antibodies. The Kabat numbering method is a scheme based on the variable regions of antibodies (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991). The EU numbering system is widely used for constant domains (including the CH1, hinge, and Fc portions) (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991).

As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to DLL3 is substantially free of antibodies that do not bind to DLL3; an isolated antibody that specifically binds to CD3 is substantially free of antibodies that do not bind to CD3; a bispecific antibody that specifically binds to CD3 and DLL3 is substantially free of antibodies that do not bind to CD3 and DLL3). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.

As used herein, the term monoclonal antibody 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. The monoclonal antibodies of the invention can be produced by a hybridoma method, phage display technology, single-gene lymphocyte cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a human light chain transgene.

As used herein, the term antigen-binding fragment refers to a fragment of an antibody such as, for example, a diabody, Fab, Fab', F(ab') ₂ , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ₂ , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single chain antibody molecule (scFv), single domain antibody (sdab), scFv dimer (bivalent diabody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camelid single domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other fragment of an antibody that binds to an antigen but does not have the complete structure of an antibody. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody or parent antibody fragment binds. According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and a heavy chain Fd segment. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab').

As used herein, the term single chain antibody refers to a conventional single chain antibody in the art that comprises a heavy chain variable region and a light chain variable region connected by a short peptide of approximately 15 to 20 amino acids. As used herein, the term single domain antibody refers to

- 13 047669 a conventional single-domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or which comprises only a heavy chain variable region.

As used herein, the term "human antibody" refers to an antibody produced by a human or to an antibody having an amino acid sequence corresponding to an antibody produced by a human, obtained using any method known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.

As used herein, the term "humanized antibody" refers to an antibody of non-human origin that has been modified to increase sequence homology with a human antibody sequence such that the antigen-binding properties of the antibody are retained but its antigenicity in the human body is reduced.

As used herein, the term chimeric antibody refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often corresponds to the variable region of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and potency, while the constant regions correspond to sequences of an antibody derived from another mammalian species (e.g., human) to avoid an immune response in that species.

As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as on different proteins (or subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth variable domain of an immunoglobulin. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. The bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, such as on different proteins (or subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises half of an antibody or fragment thereof that has binding specificity for a first epitope and half of an antibody or fragment thereof that has binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

As used herein, the term CD3 refers to cluster of differentiation 3. The exemplary amino acid sequence of the human CD3 epsilon subunit is submitted to GenBank accession number NP_000724.1. The term 4-1BB refers to tumor necrosis factor receptor superfamily member 9 (TNFRSF9), also known as CD137 and ILA (inducible by lymphocyte activation). The exemplary amino acid sequence of human 4-1BB is submitted to GenBank accession number NP_001552.2. The term 'OX40 refers to tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as CD134. The exemplary amino acid sequence

- 14 047669

Human OX40 is submitted to GenBank under accession number NP_003318.1. The term CD28 refers to cluster of differentiation 28. Exemplary amino acid sequences of human CD28 variants are submitted to GenBank under accession numbers NP_001230006.1, NP_001230007.1, NP_006130.1, XP_011510496.1, and XP_011510499.1. The term PD-1 refers to programmed cell death 1. A representative amino acid sequence of human PD-1 is submitted to GenBank under accession number NP_005009.2. The term GITR refers to glucocorticoid-inducible TNFR-related protein (GITR), also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18) or activation-inducible TNFR family receptor (AITR). Exemplary amino acid sequences of human GITR variants are submitted to GenBank under accession numbers NP_004186.1, NP_683699.1, and NP_683700.1. The term VISTA refers to V-domain Ig suppressor of T-cell activation, also known as V-set immunoregulatory receptor (VSIR). The exemplary amino acid sequence of human VISTA is submitted to GenBank under accession number NP_071436.1.

As used herein, the term "antibody that specifically binds to CD3 and/or DLL3" refers to an antibody that binds to CD3 and/or DLL3, preferably human CD3 and/or human DLL3, with a KD of 1x ^10-7 M or less, preferably 1x ^10-8 M or less, more preferably 5x ^10-9 M or less, 1x ^10-9 M or less, 5x ^10-10 M or less, or 1x ^10-10 M or less. The term KD refers to a dissociation constant that is derived from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods known in the art in light of the present invention. For example, the KD of an antibody can be determined using surface plasmon resonance, such as with a biosensor system such as the Biacore® system, or using biolayer interferometry technology such as the Octet RED96 system.

The lower the KD value of an antibody, the higher the affinity with which the antibody binds to the target antigen.

According to one particular aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH); and a light chain variable region (VL); wherein the antibody or antigen-binding fragment thereof binds to a target antigen, preferably a human target antigen; wherein an amino acid residue in the VH, VL or within twenty (20) amino acids, preferably within five (5) amino acids, of the VH or VL is replaced with an amino acid residue that is conjugated to a fatty acid (FA); and wherein when conjugated to FA on the replaced amino acid residue, the antibody or antigen-binding fragment thereof still binds to the target antigen; and wherein the antibody conjugated to FA or antigen-binding fragment thereof reduces or eliminates specific binding to a target antigen in the presence of physiological levels of albumin (e.g., 35 to 50 mg/mL). The substituted amino acid residue may, for example, be a cysteine residue or a lysine residue.

As used herein, the phrase twenty (20) amino acids away from VH or VL refers to a residue in the CH1 or CL region that is less than 20 amino acids away from the variable region of the heavy or light chain. The phrase five (5) amino acids away from VH or VL refers to a residue in the CH1 or CL region that is less than 5 amino acids away from the variable region of the heavy or light chain.

As used herein, the phrase "still binds to the target antigen" indicates that the antibody or antigen-binding fragment thereof, when conjugated to a fatty acid (FA), is still capable of binding the target antigen. The level of binding of the target antigen to the antibody conjugated to FA or its antigen-binding fragment may, for example, be from about 10% to about 100% of the level of binding of the target antigen to the antibody or its antigen-binding fragment containing the amino acid substitution for conjugation according to the invention in the absence of conjugated FA. In some embodiments, the level of binding of the target antigen to the FA-conjugated antibody or antigen-binding fragment thereof is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the level of binding of the target antigen to the antibody or antigen-binding fragment thereof comprising an amino acid substitution for conjugation of the invention in the absence of conjugated FA. One skilled in the art will be able to determine the level of binding of the FA-conjugated antibody or antigen-binding fragment thereof to the target antigen using methods known in the art. The level of binding can be compared to an antibody or antigen-binding fragment thereof comprising an amino acid substitution for conjugation of the invention that is not conjugated to a fatty acid. The level of binding of the target antigen to the antibody or antigen-binding fragment thereof containing an amino acid substitution for conjugation according to the invention, which is not conjugated to a fatty acid, is at least 50% of the level of the wild-type antibody or antigen-binding fragment thereof.

- 15 047669

According to a particular aspect, the substituted amino acid is at an amino acid residue corresponding to residue 25, 27, 62, 64, 73, 76, 101, 112 or 113 of SEQ ID NO:1, or an amino acid residue corresponding to residue 26, 27, 52, 53, 56 or 67 of SEQ ID NO:2, preferably the substitution is selected from a substitution corresponding to S25C, Y27C, K62C, K64C, K73C, S76C, D101C, S112C or S113C of SEQ ID NO:1 or a substitution corresponding to S26C, S27C, S52C, K53C, S56C or S67C of SEQ ID NO:2, wherein the residues are numbered according to Kabat. In some embodiments, the substituted amino acid is at residue 64 of SEQ ID NO:1 or at residue 26 of SEQ ID NO:2, preferably the substitution is selected from substitution K64C of SEQ ID NO:1 or substitution S26C of SEQ ID NO:2, wherein the residues are numbered according to Kabat.

As used herein, when referring to a substituted amino acid corresponding to an amino acid residue number in SEQ ID NO, SEQ ID NO is a reference for identifying the substituted amino acid residue in a sequence of interest. One skilled in the art would align the sequence of interest with the reference SEQ ID NO to determine the position of the amino acid residue to be substituted. For example, amino acid residue 25 in SEQ ID NO: 1, which is the variable region of the heavy chain of a monoclonal anti-CD3 antibody, is a serine residue. After alignment with the variable region of the heavy chain of the antibody of interest, the residue that aligns with the serine residue at position 25 of SEQ ID NO: 1 will be the target for amino group substitution.

According to a particular aspect, the substituted amino acid is at residue 119 or 120 in CH1 of SEQ ID NO:9, 10, 11 or 12, or at residue 121 or 124 in CL of SEQ ID NO:13 or 14, preferably the substitution is selected from S119C or T120C in CH1 of SEQ ID NO:9, 10, 11 or 12, or S121C or Q124C in CL of SEQ ID NO:13 or 14, wherein the residues are numbered according to EU numbering. In some embodiments, the substituted amino acid is at residue 120 in the CH1 region of SEQ ID NO:9, 10, 11 or 12, preferably the substitution is a T120C substitution in the CH1 region of SEQ ID NO:9, 10, 11 or 12, wherein the residues are numbered according to EU numbering.

According to a particular aspect, the isolated antibody or antigen-binding fragment thereof is an anti-CD3 antibody or antigen-binding fragment thereof and is capable of specifically binding to CD3, preferably human CD3. The isolated anti-CD3 antibody or antigen-binding fragment thereof may, for example, comprise a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NOs: 3, 4, 5, 6, 7 and 8, respectively, or SEQ ID NOs: 33, 34, 35, 36, 37 and 38, respectively.

According to a particular aspect, the substituted amino acid is selected from residues 25, 27, 62, 64, 73, 76, 101, 112 or 113 in the VH of the anti-CD3 mAb (SEQ ID NO:1 or SEQ ID NO:27) or 26, 27, 52, 53, 56 or 67 in the VL of the anti-CD3 mAb (SEQ ID NO:2 or SEQ ID NO:28), preferably the substitution is selected from S25C, Y27C, K62C, K64C, K73C, S76C, D101C, S112C or S113C in the VH (SEQ ID NO:1 or 27) or S26C, S27C, S52C, K53C, S56C or S67C in the VL (SEQ ID NO:2 or 28), wherein the residues are numbered according to Kabat. In some embodiments, the substituted amino acid is at residue 64 in VH (SEQ ID NO: 1 or 27) or 26 in VL (SEQ ID NO: 2 or 28), preferably the substitution is selected from a K64C substitution in VH (SEQ ID NO: 1 or 27) or a S26C substitution in VL (SEQ ID NO: 2 or 28), wherein the residues are numbered according to Kabat.

According to a particular aspect, the isolated anti-CD3 antibody or antigen-binding fragment thereof may, for example, comprise a VH region having the polypeptide sequence of SEQ ID NO:1 with the amino acid substitution K64C, and a VL region having the polypeptide sequence of SEQ ID NO:2; or a VH region having the polypeptide sequence of SEQ ID NO:27 with the amino acid substitution K64C, and a VL region having the polypeptide sequence of SEQ ID NO:28; or a VH region having the polypeptide sequence of SEQ ID NO:1 and a VL region having the polypeptide sequence of SEQ ID NO:2 with the amino acid substitution S26C; or a VH region having the polypeptide sequence of SEQ ID NO:27 and a VL region having the polypeptide sequence of SEQ ID NO:28 with the amino acid substitution S26C; or a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12, with the amino acid substitution T120C, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14; or a VH region with a polypeptide sequence of SEQ ID NO:1, a VL region with a polypeptide sequence of SEQ ID NO:2, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12 with the amino acid substitution T120C, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14; or a VH region with a polypeptide sequence of SEQ ID NO:27, a VL region with a polypeptide sequence of SEQ ID NO:28, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12 with the amino acid substitution T120C and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14.

According to a particular aspect, the present document provides a multispecific antibody or antigen-binding fragment thereof, wherein the multispecific antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof according to

- 16 047669 to the invention, and wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA. The multispecific antibody or antigen-binding fragment thereof may, for example, be a bispecific antibody or antigen-binding fragment thereof.

In some embodiments, each arm of the multispecific antibody or antigen-binding fragment thereof may comprise a substituted amino acid at a different residue position with the same or different conjugated FA. In some embodiments, each arm of the multispecific antibody or antigen-binding fragment thereof may comprise the same substituted amino acid at a different residue position with the same or different conjugated FA. In some embodiments, each arm of the multispecific antibody or antigen-binding fragment thereof may comprise a substituted amino acid at the same residue position with the same or different conjugated FA. In some embodiments, each arm of the multispecific antibody or antigen-binding fragment thereof may comprise the same substituted amino acid at the same residue position with the same or different conjugated FA.

In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) and a second antigen-binding arm (Ab2), wherein Ab 1 and/or Ab2 comprise a substituted amino acid that is conjugated to FA.

In some embodiments, Ab1 binds to an immune cell modulator (ICM), preferably a human ICM selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules. The ICM can, for example, be CD3, preferably human CD3.

In some embodiments, Ab2 binds to a tumor-associated antigen (TAA), preferably a human tumor-associated antigen (human TAA). For example, the TAA can be DLL3.

In one embodiment of the invention, an isolated bispecific antibody or antigen-binding fragment thereof of the invention is a bispecific anti-CD3/anti-DLL3 antibody or antigen-binding fragment thereof, wherein the first antigen-binding arm (Ab1) specifically binds CD3, preferably human CD3, and the second antigen-binding arm (Ab2) specifically binds DLL3, preferably human DLL3.

According to a specific aspect, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising H1 and L1, and a second antigen-binding arm (Ab2) comprising H2 and L2, wherein:

(a) H1 and H2 each comprise the CH1 region of a human IgG1, IgG2, IgG3, or IgG4 and (b) each of L1 and L2 comprises the CL region of a human kappa light chain or a human lambda light chain;

where each H1L1 and H2L2 contains a charge pair selected from the group consisting of the following amino acid substitutions:

(1) G166D/E in CH1 H1 and S114K/R in CL L1, respectively, and G166K/R in CH1 H2 and S114D/E in CL L2, respectively;

(2) T187D/E in CH1 H1 and D/N170K/R in CL L1, respectively, T187K/R in CH1 H2 and D/N170D/E in CL L2, respectively;

(3) S131D/E in CH1 H1 and P119K/R in CL L1, respectively, S131K/R in CH1 H2 and P119D/E in CL L2, respectively;

(4) A129D/E in CH1 H1 and S121K/R in CL L1, respectively, A129K/R in CH1 H2 and S121D/E in CL L2, respectively;

(5) K/R133D/E in CH1 H1 and K207K/R in CL L1, respectively, K/R133K/R in CH1 H2 and K207D/E in CL L2, respectively;

(6) K/R133D/E in CH1 H1 and I/L117K/R in CL L1, respectively, K/R133K/R in CH1 H2 and I/L117D/E in CL L2, respectively;

(7) K/R133D/E in CH1 H1 and F/V209K/R in CL L1, respectively, K/R133K/R in CH1 H2 and F/V209D/E in CL L2, respectively;

(8) G166D/E in CH1 H2 and S114K/R in CL L2, respectively, and G166K/R in CH1 H1 and S114D/E in CL L1, respectively;

(9) T187D/E in CH1 H2 and D/N170K/R in CL L2, respectively, T187K/R in CH1 H1 and D/N170D/E in CL L1, respectively;

(10) S131D/E in CH1 H2 and P119K/R in CL L2, respectively, S131K/R in CH1 H1 and P119D/E in CL L1, respectively;

(11) A129D/E in CH1 H2 and S121K/R in CL L2, respectively, A129K/R in CH1 H1 and S121D/E in CL L1, respectively;

(12) K/R133D/E in CH1 H2 and K207K/R in CL L2, respectively, K/R133K/R in CH1 H1 and K207D/E in CL L1, respectively;

- 17 047669 (13) K/R133D/E in CH1 H2 and I/L117K/R in CL L2, respectively, K/R133K/R in CH1 H1 and I/L117D/E in

CL L1, respectively; or (14) K/R133D/E in CH1 H2 and F/V209K/R in CL L2, respectively, K/R133K/R in CH1 H1 and F/V209D/E in CL L1, respectively.

As used herein, the term charge pair refers to a pair of amino acids, one of which has a positive charge and the other has a negative charge, which can be introduced by substituting native amino acid residues in the CH1 region of the heavy chain and in the CL region of the light chain of the first arm of the bispecific antibody, respectively, and at the same time the same pair of amino acids with a positive and negative charge can be introduced by substituting native amino acid residues in the CL region of the light chain and the CH1 region of the heavy chain of the second arm of the bispecific antibody, respectively. Alternatively, amino acids with a positive and negative charge can be introduced by amino acid substitution in the VH region of the heavy chain and the VL region of the light chain of the first arm of the bispecific antibody, respectively, and at the same time the same pair of amino acids with a positive charge and amino acids with a negative charge can be introduced by amino acid substitution in the VL region of the light chain and the VH region of the heavy chain of the second arm, respectively. Amino acids used to form charge pairs typically include D/E (negative charge) and K/R (positive charge). After introduction into the CH1/CL region or VH/VL region, amino acids with charge pairs are structurally close and are expected to enhance the interaction of heavy and light chains of the same arm through opposite charges and eliminate the interaction of mispaired heavy/light chains (incompatible heavy and light chains from two different arms) through like charges. The resulting charge distribution of the introduced charge pair is as follows: H1 (positive charge of CH1)/L1 (negative charge of CL)/H2 (negative charge of CH1)/L2 (positive charge of CL) or H1 (negative charge of CH1)/L1 (positive charge of CL)/H2 (positive charge of CH1)/L2 (negative charge of CL). Several charge pairs can be combined and introduced into the CH1 and CL interface, where all positively charged amino acids are introduced into CH1 and all negatively charged amino acids are introduced into CL of the same arm, or vice versa, to match the above distribution pattern. A similar approach can be applied to the VH/VL interface. In addition, one or more charge pairs may also be introduced at the VH and VL interface in combination with one or more charge pairs introduced at the CH1/CL interface - amino acids introduced into the same chain (any of H1, L1, H2 or L2) usually have the same charge, and the resulting distribution of introduced charge pairs is as follows: H1 (positive charge of CH1 and VH)/L1 (negative charge of CL and VL)/H2 (negative charge of CH1 and VH)/L2 (positive charge of CL and VL) or H1 (negative charge of CH1 and VH)/L1 (positive charge of CL and VL)/H2 (positive charge of CH1 and VH)/L2 (negative charge of CL and VL). Charge pair substitutions can also be combined with other modifications to further improve the pairing preference of the cognate chains (H1L1 and H2L2, respectively) and/or facilitate purification of the bispecific antibody using ion exchange chromatography and/or CHC. For example, in addition to charge pair substitutions, a native interchain disulfide bond on one arm of the bispecific antibody can be displaced while the other arm has a native interchain disulfide bond (see, e.g., PCT/US2020/063066, filed December 3, 2020, which is incorporated herein by reference in its entirety).

When describing charge pairs, G166D/E represents a substitution of G at position 166 (EU numbering) by D or E, in which case G166 is the knockin site; D170D/E represents retention of D at position 170 or a substitution of D at position 170 by E; K/R133D/E represents a substitution of K or R (whichever is at that position) at position 133 by D or E; all other substitutions follow the same naming convention.

According to a particular aspect, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:15, a VL region with a polypeptide sequence of SEQ ID NO:17, a CH1 region with a polypeptide sequence of SEQ ID NO:16, and a CL region with a polypeptide sequence of SEQ ID NO:18.

According to a particular aspect, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:19, a VL region with the polypeptide sequence of SEQ ID NO:21, a CH1 region with the polypeptide sequence of SEQ ID NO:20, and a CL region with the polypeptide sequence of SEQ ID NO:22.

According to a particular aspect, the bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:29, a VL region with the polypeptide sequence of SEQ ID NO:30, a CH1 region with the polypeptide sequence of SEQ ID NO:16, and a CL region with the polypeptide sequence of SEQ ID NO:18.

According to a specific aspect, the bispecific antibody or antigen-binding fragment thereof

- 18 047669 contains a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:31, a VL region with the polypeptide sequence of SEQ ID NO:32, a region

CH1 with the polypeptide sequence of SEQ ID NO:20, and the CL region with the polypeptide sequence of SEQ ID NO:22.

According to a specific aspect, the bispecific antibody or antigen-binding fragment thereof comprises:

(a) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO: 15, a VL region with a polypeptide sequence of SEQ ID NO: 17, a CH1 region with a polypeptide sequence of SEQ ID NO: 16, and a CL region with a polypeptide sequence of SEQ ID NO: 18; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO: 23, a VL region with a polypeptide sequence of SEQ ID NO: 25, a CH1 region with a polypeptide sequence of SEQ ID NO: 24, and a CL region with a polypeptide sequence of SEQ ID NO: 26;

(b) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:19, a VL region with a polypeptide sequence of SEQ ID NO:21, a CH1 region with a polypeptide sequence of SEQ ID NO:20, and a CL region with a polypeptide sequence of SEQ ID NO:22; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26;

(c) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:29, a VL region with a polypeptide sequence of SEQ ID NO:30, a CH1 region with a polypeptide sequence of SEQ ID NO:16, and a CL region with a polypeptide sequence of SEQ ID NO:18; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26; or (d) a first antigen-binding arm (Ab1) comprising a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:31, a VL region with a polypeptide sequence of SEQ ID NO:32, a CH1 region with a polypeptide sequence of SEQ ID NO:20, and a CL region with a polypeptide sequence of SEQ ID NO:22; and a second antigen-binding arm (Ab2) comprising a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26.

In some embodiments, the isolated antibody or antigen-binding fragment thereof is conjugated to an FA at a substituted amino acid residue. The FA can be, for example, selected from an FA with 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, or 18 carbon atoms. In some embodiments, the FA is selected from an FA with 14 carbon atoms or 18 carbon atoms or any number of carbon atoms therebetween. The length of the FA can determine the relative binding of albumin to the FA, which can determine the relative binding of the antibody or antigen-binding fragment thereof to the target antigen. The longer the conjugated FA, the higher the binding affinity of the conjugated FA to albumin, resulting in greater albumin-mediated reduction in the specific binding of the conjugated mAb or bsAb to the target antigen. The shorter the FA, the lower the binding affinity of the conjugated FA to albumin, resulting in less or negligible albumin-mediated reduction in the specific binding of the conjugated mAb or bsAb to the target antigen.

In some embodiments, the FA comprises a linker for conjugation with a substituted amino acid residue. The linker can be, for example, selected from a peptide linker or a polyethylene glycol (PEG) linker. The peptide linker can, for example, consist of less than 50 amino acids. The peptide linker may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 or less amino acids.

In some embodiments, FA conjugated to the antibody or antigen-binding fragment thereof is capable of binding albumin. The binding of albumin to FA results in a partial or complete block of binding of the target antigen to the antibody or antigen-binding fragment thereof. In some embodiments, where the isolated antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, wherein only the Ab1 arm is conjugated to FA, the binding of albumin to FA does not affect the binding of the Ab2 arm to TAA. In some embodiments, wherein the isolated antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, wherein both arms of Ab1 and Ab2 are conjugated to FA, the binding of albumin to FA results in a decrease or elimination of the binding of Ab1 and Ab2 to the target antigen of Ab1 and Ab2, respectively. In some embodiments, the isolated antibody or antigen-binding fragment thereof has a reduced ability to activate T cells upon binding to albumin, compared to an isolated antibody or antigen-binding fragment thereof that does not bind to albumin.

In one embodiment of the invention, the anti-CD3/anti-DLL3 bispecific antibody or antigen-binding fragment thereof of the invention is capable of activating T cells.

The full-length bispecific antibodies of the present invention can be prepared, for example, using Fab arm swapping (or half-molecule swapping) between two monospecific divalent antibodies by introducing substitutions at the CH3 heavy chain interface in each half molecule to promote the formation of a heterodimer of the two half antibody molecules having distinct specificities either in vitro in a cell-free medium or by co-expression. The Fab arm swap reaction is a result of a disulfide bond isomerization reaction and a dissociation-association of the CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parent monospecific antibodies are reduced. The resulting free cysteines of one of the parent monospecific antibodies form a heavy chain disulfide bond with the cysteine residues of the second parent monospecific antibody molecule, and the CH3 domains of the parent antibodies are simultaneously released and restored by dissociation-association. The CH3 domains of the Fab arms can be engineered to favor heterodimerization rather than homodimerization. The resulting product is a bispecific antibody having two Fab arms or half-molecules, each binding a distinct epitope, i.e., an epitope on CD3 and an epitope on DLL3.

As used herein, the term homodimerization refers to the interaction of two heavy chains having identical CH3 amino acid sequences. As used herein, the term homodimer refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

As used herein, the term heterodimerization refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. As used herein, the term heterodimer refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

The bulge-in-hole strategy (see, e.g., PCT Publication No. WO 2006/028936) can be used to generate full-length bispecific antibodies. Briefly, selected amino acids forming the interface between the CH3 domains of human IgG can be mutated at positions affecting the interaction of the CH3 domains to promote the formation of a heterodimer. An amino acid with a small side chain (the hole) is introduced into the heavy chain of the antibody specifically binding to the first antigen, and an amino acid with a large side chain (the bulge) is introduced into the heavy chain of the antibody specifically binding to the second antigen. Upon coexpression of the two antibodies, a heterodimer is formed by the preferential interaction of the hole heavy chain with the bulge heavy chain. Typical CH3 substitution pairs that form a bulge and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S, and T366W/T366S_L368A_Y407V.

Other strategies may be used, such as promoting heavy chain heterodimerization using electrostatic interactions by exchanging positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publication No. US2010/0015133; U.S. Patent Publication No. US2009/0182127; U.S. Patent Publication No. US2010/028637; or U.S. Patent Publication No. US2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V K409F Y407A/T366A_K409F or T350V_L351Y_F405A Y407V/T350V_T366L_K392L_T394W, as described in U.S. Patent No. US2012/0149876 or U.S. Patent Publication No. US2013/0195849.

In addition to the methods described above, the bispecific antibodies of the invention can be produced in vitro in a cell-free medium by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions that allow isomerization of the disulfide bond, in accordance with the methods described in PCT Patent Publication No. WO2011/131746. In the methods, the first monospecific bivalent antibody and the second monospecific bivalent antibody are designed to have specific

- 20 047 669 substitutions in the CH3 domain that contribute to the stability of the heterodimer; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby creating a bispecific antibody by exchanging Fab arms. The incubation conditions can optionally be reduced to non-reducing conditions. Examples of reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris-(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably the reducing agent is selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris-(2-carboxyethyl)phosphine. For example, incubation for at least 90 min at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of 5 to 8, such as pH 7.0 or pH 7.4, can be used.

Full-length bispecific antibodies of the invention can be prepared using a combination of the heterodimerization approaches described above and more than one of the following approaches: (a) shifting the HC/LC interchain disulfide bond on one arm of the bispecific antibody (see, e.g., PCT/US2020/063066, filed December 3, 2020, which is incorporated herein by reference in its entirety); (b) introducing charge pairs into the VH/VL interface; (c) introducing charge pairs into the CH1/CL interface; or (d) a combination of some or all of the approaches described in (a)-(c) (see, e.g., as first described in U.S. Provisional Patent Application No. 63/146,334, filed February 5, 2021, which is incorporated herein by reference in its entirety).

In another general aspect, the invention relates to an isolated nucleic acid encoding an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the invention. Those skilled in the art will appreciate that the coding sequence of a protein can be altered (e.g., replaced, deleted, inserted, etc.) without altering the amino acid sequence of the protein. Accordingly, those skilled in the art will appreciate that nucleic acid sequences encoding the antibodies or antigen-binding fragments thereof of the invention can be altered without altering the amino acid sequences of the proteins.

In another general aspect, the invention provides a vector comprising an isolated nucleic acid encoding an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the invention. Any vector known to those skilled in the art in light of the present invention can be used, such as a plasmid, a cosmid, a phage vector, or a viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the normal function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids into a cell are known in the art and can be used herein to produce an antibody or antigen-binding fragment thereof in a cell. Conventional cloning techniques or artificial gene synthesis may be used to create a recombinant expression vector according to embodiments of the invention. Such techniques are well known to those skilled in the art in connection with the present disclosure.

In another general aspect, the invention provides a host cell comprising a vector comprising an isolated nucleic acid encoding an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof, of the invention. Any host cell known to those skilled in the art in connection with the present disclosure can be used to recombinantly express the antibodies or antigen-binding fragments thereof of the invention. In some embodiments, the host cells are E. coli TG1 or BL21 cells (for expressing, e.g., an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, e.g., a full-length IgG antibody). According to certain embodiments, the recombinant expression vector is transformed into host cells by conventional methods, such as chemical transfection, heat shock or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is efficiently expressed.

In another general aspect, the invention relates to a method for producing an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the present invention, comprising culturing a cell containing a nucleic acid encoding the antibody or antigen-binding fragment thereof under conditions that allow production of the antibody or antigen-binding fragment thereof of the present invention, and isolating the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from a supernatant). The expressed antibodies or antigen-binding fragments thereof can be collected from the cells and purified according to conventional methods,

- 21 047669 known in the art and as described herein.

In another general aspect, the invention relates to a method for producing an isolated antibody or antigen-binding fragment thereof conjugated to an FA of the invention. The methods comprise conjugating FA to the antibody or antigen-binding fragment thereof at a substituted amino acid residue and isolating the antibody or antigen-binding fragment thereof conjugated to the FA.

In another general aspect, the invention relates to a method for producing an isolated antibody or antigen-binding fragment thereof conjugated to FA and bound to albumin. The methods comprise contacting the isolated antibody or antigen-binding fragment thereof conjugated to FA with albumin and isolating the antibody or antigen-binding fragment thereof conjugated to FA bound to albumin.

Pharmaceutical compositions.

In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier. The isolated monoclonal or bispecific antibody or antigen-binding fragment thereof can, for example, be conjugated to a fatty acid (FA). The FA-conjugated monoclonal or bispecific antibody or antigen-binding fragment thereof can, for example, bind to albumin. As used herein, the term pharmaceutical composition means a product comprising an antibody of the invention together with a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions comprising them can also be used in the manufacture of the medicament for therapeutic use mentioned herein.

As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other material well known in the art for use in pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the composition of the invention or the biological activity of the composition of the invention. According to certain embodiments, given the present disclosure, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical composition of an antibody can be used in the invention.

The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any later editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity adjusting agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the invention.

In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation containing water. The liquid formulation may include a solution, a suspension, an emulsion, a microemulsion, a gel, and the like. The aqueous formulation typically contains at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w/w water.

In one embodiment, the pharmaceutical composition may be formulated as an injection preparation that can be administered, for example, using an injection device (e.g., a syringe or an infusion pump). The injection can be administered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

In another embodiment, the pharmaceutical composition is a solid composition, such as a lyophilized or spray-dried composition, which can be used as is or to which solvents and/or diluents are added by the physician or patient prior to use. Solid dosage forms may include tablets, such as compressed and/or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The pharmaceutical composition may also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.

Dosage forms may be immediate release, in which case they may include a water-soluble or dispersible carrier, or they may be delayed release, extended release, or modified release, in which case they may contain water-insoluble polymers that control the rate of dissolution of the dosage form in the gastrointestinal tract or under the skin.

In other embodiments, the pharmaceutical composition can be administered intranasally, intrabuccally, or sublingually.

The pH of the aqueous composition may be between pH 3 and pH 10. In one embodiment of the invention,

- 22 047669 the pH of the composition is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the composition is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris-(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer may be present individually or in aggregate at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal and mixtures thereof. The preservative may be present individually or in combination at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific preservatives represent alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of isotonic agents include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), an alditol (such as glycerol, 1,2-propanediol, propylene glycol), 1,3-propanediol and 1,3-butanediol), polyethylene glycol (e.g., PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars can be mono-, di- or polysaccharides or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha- and beta-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, where the term sugar alcohol is defined as a C(48) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol and arabitol. The isotonic agent may be present individually or in combination at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these particular isotonic agents are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agent may be present individually or in combination at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions comprising each of these specific chelating agents are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy/hydroxycellulose and derivatives thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethyleneglycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances such as monothioglycerol) or thioglycolic acid. The stabilizer may be present individually or in combination at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific stabilizers are alternative embodiments of the invention.

In further embodiments of the invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant or two different surfactants. The term surfactant refers to any molecule or ion that consists of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants,

- 23 047 669 substances and/or zwitterionic surfactants. The surfactant may be present individually or in combination in a concentration of from about 0.1 mg/mL to about mg/mL. Pharmaceutical compositions containing each of these specific surfactants are alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and/or benzamidine hydrochloric acid (HCl). The protease inhibitor may be present individually or in combination at a concentration of from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific protease inhibitors are alternative embodiments of the invention.

In another general aspect, the invention relates to a method for producing a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof according to the invention, comprising combining the antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to produce the pharmaceutical composition.

Methods of application.

In another general aspect, the invention relates to a method of targeting a tumor-associated antigen (TAA) (e.g., DLL3) expressed on the surface of cancer cells in a subject in need thereof. The methods comprise administering to the subject a pharmaceutical composition comprising an isolated bispecific antibody or antigen-binding fragment thereof comprising an Ab1 arm (e.g., an anti-ICM arm, such as an anti-CD3 arm) conjugated to FA (e.g., an anti-CD3/anti-DLL3 bispecific antibody or antigen-binding fragment thereof) of the invention and a pharmaceutically acceptable carrier. Simultaneous binding of the isolated FA-conjugated bispecific antibody or antigen-binding fragment thereof to a TAA-expressing cancer cell via the anti-TAA arm (Ab2 arm) and a T cell via the anti-CD3 arm (Ab1 arm), at low albumin levels, can promote the killing of cancer cells. In circulating blood, where albumin levels are high (e.g., 35 to 50 mg/mL), the FA-conjugated anti-CD3 arm is in an albumin-bound state and therefore has a reduced ability to bind and activate T cells. The target T cell antigen to which the Ab1 arm binds may be another T cell ICM such as 4-1BB, GITR, CD28, or PD-1. This approach may increase the safety margin of an anti-ICM (e.g., anti-CD3)-based bispecific T cell attractor by minimizing on-target and extra-tumor toxicity. Additionally, this approach may be applicable to bsAbs (including the anti-TAA arm and the conjugated anti-ICM arm), which may be used as interfering agents with other immune cells. Furthermore, this approach could be applied to the application of FA-conjugated mAbs and/or bsAbs to target tissues (such as adipose tissue or skeletal muscle) where local albumin levels are lower than in circulating blood to minimize target safety concerns in the circulation (Ellmerer et al., Am J Physiol Endocrinol Metab. 2000. 278: E352-E356).

The functional activity of monoclonal antibodies or antigen-binding fragments thereof that bind a target antigen (e.g., ICM, such as CD3), or bispecific antibodies and antigen-binding fragments thereof that bind both TAA (e.g., DLL3) and a target T cell antigen (e.g., ICM, such as CD3) can be characterized by methods known in the art and described herein. Methods for characterizing bispecific antibodies and antigen-binding fragments thereof that bind both TAA (e.g., DLL3) and a target T cell antigen (e.g., CD3) include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, FACS, and OctetRed assays. According to particular embodiments, methods for characterizing bispecific antibodies and antigen-binding fragments thereof that bind both DLL3 and CD3 include the methods described below. The functional activity of monoclonal antibodies or antigen-binding fragments thereof that bind ICM, or bispecific antibodies and antigen-binding fragments thereof that bind both TAA (e.g., DLL3) and ICM other than CD3, can be characterized by methods similar to those described above.

In another general aspect, the invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject in need thereof an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the invention. The cancer may be any hematological malignancy or solid cancer, for example, it may be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic

- 24 047669 lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other hemoblastoses.

According to embodiments of the invention, the pharmaceutical composition comprises a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention. As used herein, the term therapeutically effective amount refers to the amount of an active ingredient or component that causes a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and in a routine manner depending on the stated purpose.

As used herein with respect to monoclonal and/or bispecific antibodies or antigen-binding fragments thereof, a therapeutically effective amount means an amount of a monoclonal and/or bispecific antibody or antigen-binding fragment thereof that modulates an immune response in a subject in need thereof. Also as used herein with respect to monoclonal and/or bispecific antibodies or antigen-binding fragments thereof, a therapeutically effective amount means an amount of a monoclonal and/or bispecific antibody or antigen-binding fragment thereof that results in treatment of a disease, disorder, or condition; prevents or slows the progression of a disease, disorder, or condition; or reduces or alleviates symptoms associated with a disease, disorder, or condition.

According to specific embodiments, the disease, disorder or condition to be treated is a cancer, preferably a cancer selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and others. hematological malignancies. According to other specific embodiments, the disease, disorder or condition to be treated is an inflammatory disease, a metabolic disease, a cardiovascular disease, a neurological disease, an infectious disease or any other disease for which the bispecific antibody can be used as a therapy.

According to specific embodiments, a therapeutically effective amount refers to an amount of therapy sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of the disease, disorder, or condition being treated, or a symptom associated therewith; (ii) decreasing the duration of the disease, disorder, or condition being treated, or a symptom associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or a symptom associated therewith; (iv) regression of the disease, disorder, or condition being treated, or a symptom associated therewith; (v) preventing the development or onset of the disease, disorder, or condition being treated, or a symptom associated therewith; (vi) preventing the recurrence of the disease, disorder, or condition being treated, or a symptom associated therewith; (vii) reducing hospitalizations in a subject having the disease, disorder, or condition being treated, or a symptom associated therewith; (viii) decreasing the length of hospitalization of a subject with the disease, disorder, or condition being treated or a symptom associated therewith; (ix) increasing the survival of a subject with the disease, disorder, or condition being treated or a symptom associated therewith; (xi) inhibiting or reducing the disease, disorder, or condition being treated or a symptom associated therewith in a subject; and/or (xii) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

A therapeutically effective amount or dosage may vary depending on a variety of factors, such as the disease, disorder or condition being treated, the route of administration, the target site, the physiological state of the subject (including, for example, age, body weight, health), whether the subject is a human or an animal, whether other drugs are being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic doses are optimally titrated to optimize safety and efficacy.

According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

As used herein, the terms treat, cure, and treatment are intended to mean improvement in or change in at least one measurable physical parameter associated with cancer that is not necessarily discernible in the subject, but may be discernible in the subject.

- 25 047669

The terms treat, treat and treat may also refer to causing regression, preventing progression or at least slowing the progression of a disease, disorder or condition. In a particular embodiment, treat, treat and treat refer to alleviating, preventing the development or occurrence of, or shortening the duration of one or more symptoms associated with a disease, disorder or condition, such as a tumor or, more preferably, cancer. In a particular embodiment, treat, treat and treat refer to preventing the recurrence of a disease, disorder or condition. In a particular embodiment, treat, treat and treat refer to increasing the survival of a subject suffering from a disease, disorder or condition. In a particular embodiment, treat, treat and treat refer to eliminating a disease, disorder or condition in a subject.

According to specific embodiments, compositions useful for treating cancer are provided. For cancer therapy, the compositions can be used in combination with other treatments, including, but not limited to, chemotherapy, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-CD47 mAb, anti-apelin mAb, anti-CTLA-4 mAb, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD20 mAb, anti-CD33 mAb, anti-TIP-1 mAb, anti-DLL3 mAb, anti-CLDN18.2 mAb, anti-PD-L1 antibody, anti-PD-1 antibody, PD-1/PD-L1 therapy, other immuno-oncology drugs, an antiangiogenic agent, radiation therapy, an antibody-drug conjugate (ADC), targeted therapy, or other anticancer drugs.

As used herein, the term in combination in the context of administering two or more therapies to a subject refers to the use of more than one therapy. Use of the term in combination does not limit the order in which the therapeutic agents are administered to the subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of a second therapy to a subject.

Also provided are methods comprising contacting albumin with a conjugate comprising a fatty acid (FA) covalently linked, optionally via a linker, to an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof in the conjugate is capable of specifically binding to a target antigen, the FA in the conjugate is capable of binding to albumin, and the binding of albumin to FA results in a partial or complete block of binding of the target antigen to the antibody or antigen-binding fragment thereof. In some embodiments, the contacting step comprises administering a pharmaceutical composition comprising the conjugate to a subject in need of treatment of a tumor, wherein the tumor comprises a target antigen. In some embodiments, albumin has a higher metabolism in the tumor microenvironment compared to circulating blood and/or is present in the tumor microenvironment at a level lower than the level of albumin in the circulating blood of the subject.

Implementation options

The invention also provides the following non-limiting embodiments.

Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

a) variable region of the heavy chain (VH);

b) variable region of the light chain (VL);

wherein the antibody or antigen-binding fragment thereof binds to a target antigen, preferably a human target antigen;

wherein an amino acid residue in VH, VL or within twenty (20) amino acids of VH or VL on one or both arms is replaced by an amino acid residue that is conjugated to a fatty acid (FA);

and where, when conjugated to FA at the replaced amino acid residue, the antibody or antigen-binding fragment thereof still binds to the target antigen.

Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 1, wherein the replaced amino acid residue is within five (5) amino acid residues of VH or VL on one or both arms.

Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein the substituted amino acid residue is a cysteine residue, a lysine residue, or a modified amino acid suitable for chemical conjugation.

Embodiment 4 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 3, wherein the substituted amino acid residue is at an amino acid residue corresponding to:

- 26 047669 (1) remainder 25, 27, 62, 64, 73, 76, 101, 112 or 113 SEQ ID NO:1 in VH (Kabat numbering);

(2) residue 26, 27, 52, 53, 56 or 67 of SEQ ID NO:2 in VL (Kabat numbering);

(3) residue 119 or 120 of SEQ ID NO:9, 10, 11 or 12 in CH1 (EU numbering) or (4) residue 121 or 124 of SEQ ID NO:13 or 14 in CL (EU numbering).

Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 4, wherein the substituted amino acid residue is at an amino acid residue corresponding to:

(1) replacing K64C SEQ ID NO:1 in VH;

(2) replacing S26C SEQ ID NO:2 in VL or (3) replacing T120C SEQ ID NO:9, 10, 11 or 12 in CH1 region.

Embodiment 6 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1-5, wherein the antibody or antigen-binding fragment thereof is an anti-immune cell modulator (ICM) antibody or antigen-binding fragment thereof and is capable of specifically binding to ICM, preferably to human ICM.

Embodiment 7 is an isolated monoclonal antibody or antigen-binding fragment thereof according to embodiment 6, wherein the ICM is selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules.

Embodiment 8 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 7, wherein the ICM is CD3 and the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively; or SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively.

Embodiment 9 is an isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the ICM is CD3, and wherein the substituted amino acid residue is at an amino acid residue selected from:

(1) residue 25, 27, 62, 64, 73, 76, 101, 112, or 113 of SEQ ID NO:1 or 27 in VH (Kabat numbering);

(2) residue 26, 27, 52, 53, 56, or 67 of SEQ ID NO:2 or 28 in VL (Kabat numbering);

(3) residue 119 or 120 of SEQ ID NO:9, 10, 11 or 12 in CH1 (EU numbering) or (4) residue 121 or 124 of SEQ ID NO:13 or 14 in CL (EU numbering).

Embodiment 10 is an isolated monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 5-9, comprising:

(1) a VH region with a polypeptide sequence of SEQ ID NO:1 with an amino acid substitution of K64C, and a VL region with a polypeptide sequence of SEQ ID NO:2;

(2) a VH region with the polypeptide sequence of SEQ ID NO:27 with the amino acid substitution K64C, and a VL region with the polypeptide sequence of SEQ ID NO:28;

(3) a VH region with the polypeptide sequence of SEQ ID NO:1, and a VL region with the polypeptide sequence of SEQ ID NO:2 with the amino acid substitution S26C;

(4) a VH region with the polypeptide sequence of SEQ ID NO:27, and a VL region with the polypeptide sequence of SEQ ID NO:28 with the amino acid substitution S26C;

(5) a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12, with an amino acid substitution T120C, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14;

(6) a VH region with a polypeptide sequence of SEQ ID NO:1, a VL region with a polypeptide sequence of SEQ ID NO:2, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11, or 12 with the T120C amino acid substitution, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14; or (7) a VH region with a polypeptide sequence of SEQ ID NO:27, a VL region with a polypeptide sequence of SEQ ID NO:28, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11, or 12 with the T120C amino acid substitution, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14.

Embodiment 11 is an isolated multispecific antibody or antigen-binding fragment thereof, wherein the multispecific antibody or antigen-binding fragment thereof comprises a monoclonal antibody or antigen-binding fragment thereof according to any one of embodiments 1-10, and wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA.

Embodiment 12 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 11, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding arm (Ab1) and a second antigen-binding arm (Ab2), wherein Ab1 and/or Ab2 comprise a substituted amino acid that is conjugated to FA.

Embodiment 13 is an isolated bispecific antibody or antigen-binding fragment thereof of embodiment 12, wherein Ab1 binds an immune cell modulator (ICM), preferably human ICM.

Embodiment 14 is an isolated bispecific antibody or antigen-binding fragment thereof according to embodiment 13, wherein the ICM is selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules.

Embodiment 15 is an isolated bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 12-14, wherein Ab2 binds a tumor-associated antigen (TAA), preferably a human tumor-associated antigen (human TAA).

Embodiment 16 is an isolated bispecific antibody or antigen-binding fragment thereof of embodiment 15, wherein the TAA is DLL3.

Embodiment 17 is an isolated bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 12-16, wherein the first antigen-binding arm (Ab1) comprises H1 and L1, and the second antigen-binding arm (Ab2) comprises H2 and L2, wherein:

(a) H1 and H2 each comprise a CH1 region of a human IgG1, IgG2, IgG3, or IgG4; and (b) each of L1 and L2 comprises a CL region of a human kappa light chain or a human lambda light chain;

where each H1L1 and H2L2 contains a charge pair selected from the group consisting of the following amino acid substitutions:

(1) G166D/E in CH1 H1 and S114K/R in CL L1, respectively, and G166K/R in CH1 H2 and S114D/E in CL L2, respectively;

(2) T187D/E in CH1 H1 and D/N170K/R in CL L1, respectively, T187K/R in CH1 H2 and D/N170D/E in CL L2, respectively;

(3) S131D/E in CH1 H1 and P119K/R in CL L1, respectively, S131K/R in CH1 H2 and P119D/E in CL L2, respectively;

(4) A129D/E in CH1 H1 and S121K/R in CL L1, respectively, A129K/R in CH1 H2 and S121D/E in CL L2, respectively;

(5) K/R133D/E in CH1 H1 and K207K/R in CL L1, respectively, K/R133K/R in CH1 H2 and K207D/E in CL L2, respectively;

(6) K/R133D/E in CH1 H1 and I/L117K/R in CL L1, respectively, K/R133K/R in CH1 H2 and I/L117D/E in CL L2, respectively;

(7) K/R133D/E in CH1 H1 and F/V209K/R in CL L1, respectively, K/R133K/R in CH1 H2 and F/V209D/E in CL L2, respectively;

(8) G166D/E in CH1 H2 and S114K/R in CL L2, respectively, and G166K/R in CH1 H1 and S114D/E in CL L1, respectively;

(9) T187D/E in CH1 H2 and D/N170K/R in CL L2, respectively, T187K/R in CH1 H1 and D/N170D/E in CL L1, respectively;

(10) S131D/E in CH1 H2 and P119K/R in CL L2, respectively, S131K/R in CH1 H1 and P119D/E in CL L1, respectively;

(11) A129D/E in CH1 H2 and S121K/R in CL L2, respectively, A129K/R in CH1 H1 and S121D/E in CL L1, respectively;

(12) K/R133D/E in CH1 H2 and K207K/R in CL L2, respectively, K/R133K/R in CH1 H1 and K207D/E in CL L1, respectively;

(13) K/R133D/E in CH1 H2 and I/L117K/R in CL L2, respectively, K/R133K/R in CH1 H1 and I/L117D/E in CL L1, respectively; or (14) K/R133D/E in CH1 H2 and F/V209K/R in CL L2, respectively, K/R133K/R in CH1 H1 and F/V209D/E in CL L1, respectively.

Embodiment 18 is an isolated bispecific antibody or antigen-binding fragment thereof according to any one of embodiments 12-17, wherein the bispecific antibody or antigen-binding fragment thereof comprises:

a) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:15, a VL region with a polypeptide sequence of SEQ ID NO:17, a CH1 region with a polypeptide sequence of SEQ ID NO:16, and a CL region with a polypeptide sequence of SEQ ID NO:18;

b) the first antigen-binding arm (Ab1), containing the VH region with the polypeptide sequence

- 28 047669 with the polypeptide sequence SEQ ID NO:19, the VL region with the polypeptide sequence SEQ ID NO:21, the region

CH1 with the polypeptide sequence of SEQ ID NO:20, and the CL region with the polypeptide sequence of SEQ ID NO:22;

c) a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:29, a VL region with a polypeptide sequence of SEQ ID NO:30, a CH1 region with a polypeptide sequence of SEQ ID NO:16, and a CL region with a polypeptide sequence of SEQ ID NO:18; or

d) a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:31, a VL region with the polypeptide sequence of SEQ ID NO:32, a CH1 region with the polypeptide sequence of SEQ ID NO:20, and a CL region with the polypeptide sequence of SEQ ID NO:22.

Embodiment 19 is an isolated bispecific antibody or antigen-binding fragment thereof according to embodiment 18, wherein the second antigen-binding arm (Ab2) comprises a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26.

Embodiment 20 is an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-19, wherein FA is selected from FA with 6 carbon atoms, 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms, or 18 carbon atoms or any number of carbon atoms therebetween.

Embodiment 21 is an isolated antibody or antigen-binding fragment thereof of embodiment 20, wherein FA is selected from FA with 14 carbon atoms or 18 carbon atoms or any number of carbon atoms in between.

Embodiment 22 is an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-21, wherein the FA comprises a linker for conjugation to a substituted amino acid residue.

Embodiment 23 is an isolated antibody or antigen-binding fragment thereof of embodiment 22, wherein the linker is selected from a peptide linker or a polyethyleneglycol linker.

Embodiment 24 is an isolated antibody or antigen-binding fragment thereof of embodiment 23, wherein the peptide linker is less than 50 amino acids long.

Embodiment 25 is an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-24, wherein FA conjugated to the antibody or antigen-binding fragment thereof is capable of binding albumin, wherein the binding of albumin to FA results in partial or complete blocking of binding of the target antigen to the antibody or antigen-binding fragment thereof.

Embodiment 26 is an isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-25, wherein the isolated antibody or antigen-binding fragment thereof has a reduced ability to activate T cells when bound to albumin compared to an isolated antibody or antigen-binding fragment thereof not bound to albumin.

Embodiment 27 is an isolated nucleic acid encoding an isolated antibody or antigen-binding fragment thereof of any one of embodiment 126.

Embodiment 28 is a vector comprising the isolated nucleic acid of embodiment 27.

Embodiment 29 is an isolated host cell comprising the vector of embodiment 27.

Embodiment 30 is a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-26 and a pharmaceutically acceptable carrier.

Embodiment 31 is a method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of embodiment 30.

Embodiment 32 is a method according to embodiment 31, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other hematological malignancies.

Embodiment 33 is a method for producing an isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1-26, comprising culturing a cell containing a nucleic acid encoding the antibody or antigen-binding fragment thereof under conditions for producing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the cell or culture.

Embodiment 34 is the method of embodiment 33 further comprising conjugating FA to the antibody or antigen-binding fragment thereof at the substituted amino acid residue.

Embodiment 35 is a method of producing a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof, wherein the method comprises combining the antibody or antigen-binding fragment thereof of any one of embodiment 126 with a pharmaceutically acceptable carrier to produce the pharmaceutical composition.

Embodiment 36 is a method comprising contacting albumin with an isolated antibody or antigen-binding fragment thereof of any one of embodiments 1-26, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to a target antigen, FA is capable of binding to albumin, and the binding of albumin to FA results in partial or complete blocking of binding of the target antigen to the antibody or antigen-binding fragment thereof.

Embodiment 37 is the method of embodiment 36, wherein the contacting step comprises administering a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof to a subject in need of treatment of a tumor, wherein the tumor comprises the target antigen.

Embodiment 38 is the method of embodiment 36 or 37, wherein the albumin has a higher metabolism in the tumor microenvironment compared to circulating blood and/or is present in the tumor microenvironment at a level lower than the level of albumin in the circulating blood of the subject, preferably the lower level of albumin in the tumor microenvironment is due to high catabolism of albumin in the tumor microenvironment and/or high levels of proteases in the tumor microenvironment.

Examples

Example 1. Construction and characterization of monoclonal antibodies for conjugation with fatty acid molecules.

Fig. 1A schematically illustrates a monoclonal antibody (mAb) in which a residue in the VH region is identified and replaced with a cysteine (knocked-on cysteine). A fatty acid (FA) molecule containing a linker and a reactive group is conjugated to the knocked-on cysteine such that each mAb contains two FA molecules (Fig. 1A). The monoclonal antibody may also contain a replaced amino acid residue in the VL or at a distance of twenty (20) amino acids, preferably five (5) amino acids, from the VH or VL in the CH1 or CL region. The knocked-on cysteine residue may also be another reactive amino acid residue suitable for conjugation of FA.

Conjugated FA molecules can bind to albumin circulating in the blood and/or interstitial fluid in tissues. Bound albumin molecules are expected to partially or completely block the interaction of the antigen-binding domain (containing VH and VL) of the conjugated mAb with the antigen due to the steric hindrance effect of bound albumin. Therefore, the antigen-binding activity of conjugated mAbs can be regulated by the level of surrounding albumin. Depending on the location of the inserted amino acid residue, the length of the FA, the presence of a linker, and the length of the linker, different degrees of modulation of mAb binding to the target antigen can be achieved.

The mAb in Fig. 1A may, for example, be an anti-CD3 antibody. Following conjugation, the activity of the anti-CD3 mAb can be modulated in vivo by albumin such that the anti-CD3 mAb becomes inactive or less active in the circulating blood. In the tumor microenvironment, due to the higher turnover of albumin compared to the circulating blood, the concentration of unbound conjugated mAb (i.e., non-albumin-bound conjugated anti-CD3 mAb) increases and T cell activation results in the anti-CD3 mAb-mediated cancer killing effect. The mAb may be directed to other cancer targets, particularly ICM (e.g., 4-1BB, GITR, OX40, CD28, or PD-1), where the therapeutic approach requires less or no mAb activity in the circulating blood and greater activity in the tumor microenvironment. In addition, the conjugation and modulation strategy can be used with an antigen-binding fragment that is not a mAb. In this case, the FA conjugation site, the length of the FA, the presence of a linker, and the length of the linker are chosen such that the albumin bound to the FA protrudes at the interface between the antigen-binding domain and the target antigen.

A conjugated mAb or antigen-binding fragment thereof can be used to construct a bispecific antibody (bsAb) or antigen-binding fragment thereof with another antibody or antigen-binding fragment thereof. For illustrative purposes, conjugated bispecific

- 30 047669 bispecific antibodies are shown in Fig. 1B, 1C. The Ab1 arm is derived from an aHTu-CD3 antibody and the Ab2 arm is derived from an anti-tumor-associated antigen (TAA) mAb. Conjugation of an FA molecule to a region (e.g., VH, VL, or within twenty (20) amino acids, preferably five (5) amino acids, of the VH or VL region) of the anti-CD3 arm can modulate anti-CD3 activity and hence modulate T cell activation by the bispecific antibody, while binding of the anti-TAA arm to TAA is independent of surrounding albumin concentrations. The FA-conjugated bispecific antibody is expected to be less active or not active at all in circulating blood and/or some tissue fluids at high albumin levels (e.g., 35 to 50 mg/mL). The FA-conjugated bispecific antibody is expected to be active in stimulating T cells in specific tumor microenvironments due to high albumin turnover, resulting in lower local albumin levels and higher concentrations of naked conjugated bsAb (i.e., non-albumin-conjugated anti-CD3 bsAb) and enhanced cancer cell killing via T cell activation. The Ahtu-CD3 arm of the bsAb may be directed to other cancer targets, particularly ICM (e.g., 4-1BB, GITR, OX40, CD28, or PD-1), where the therapeutic approach requires less or no bsAb activity in the circulation and greater activity in the tumor microenvironment. This approach may increase the safety margin of the anti-CD3-based bispecific T cell attractor by minimizing on-target and off-tumor toxicity. Such a therapeutic agent may reduce the risk of cytokine storm syndrome (CRS), commonly seen with anti-CD3 T cell involvement. The conjugation and modulation strategy can be used with a bispecific antigen-binding fragment that is not a bispecific antibody. In this case, the FA conjugation site, the length of the FA, the presence of a linker, and the length of the linker are chosen such that the albumin bound to the FA protrudes at the interface between the antigen-binding domain and the target antigen.

Fig. 1D is a schematic showing the mechanism of action of an FA-conjugated bispecific antibody that engages T cells and kills a cancer cell. The anti-TAA arm binds to TAA on the surface of cancer cells regardless of the level of surrounding albumin. The FA-conjugated T cell-interacting arm (e.g., the anti-CD3 arm) does not bind to the target antigen (e.g., CD3) when the level of surrounding albumin is high (e.g., 35 to 50 mg/mL, such as in circulating blood); however, when the level of surrounding albumin is low, the FA-conjugated T cell-interacting arm (e.g., the anti-CD3 arm) binds to the target antigen (e.g., CD3) and activates the T cell, resulting in cancer cell death. The FA-conjugated arm can be a T cell-interacting arm against other T cell ICMs such as 4-1BB, GITR, OX40, CD28, PD-1, or any other targets that are expressed on T cells and can mediate T cell activation upon binding by a specific antibody. Additionally, the FA-conjugated arm can be directed against ICMs on other immune cells. The approach of using lower albumin levels at the target site compared to those in the circulating blood can also be applied to therapies targeting tissues with low local albumin levels; these tissues include adipose tissue and skeletal muscle (Ellmerer et al., Am J Physiol Endocrinol Metab. 2000. 278: E352-E356). In Fig. 1E shows specific steps for identifying FA-conjugated mAbs or bsAbs.

The modified anti-CD3 antibody was used to construct the conjugated mAbs. The sequences and numbering of the VH and VL regions of the anti-CD3 mAbs are shown in Figs. 2A-2B and Table 1 (SEQ ID NOs: 1 and 2, respectively). The sequences of the CDR regions (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) are listed in Table 2 (SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, and SEQ ID NOs: 33, 34, 35, 36, 37, and 38, respectively). The sequences and numbering of the CH1 regions of the heavy chains (HC) of IgG1, IgG2, IgG3, and IgG4 are shown in Fig. 2C and Table 1 (SeQ ID NOs: 9, 10, 11, and 12, respectively). The sequences and numbering of the CL regions of the kappa and lambda light chains (LC) are shown in Fig. 2D and Table 1 (SEQ ID NOs:13 and 14, respectively).

Residues on the surface of the Fab region of the anti-CD3 antibody were identified by structural modeling. The anti-CD3 mAb sequence was modeled as 1SY6 and 3EO9 using Schrodinger Bioluminate® (Schrodinger; New York, NY). Side chain accessibility was calculated, and residues in or near the variable regions of both the heavy and light chains with side chain accessibility in the range of 30% to 70% were selected as possible candidates for cysteine incorporation. The residues identified for knockin are listed in Table 3. The four residues selected for the cysteine knockin experiment are shown in the three-dimensional structure of the anti-CD3 mAb as examples (Fig. 3A): S26 and S31 in the VL region, K64 in the VH region, and T120 in the CH1 region of the HC (3 amino acid residues from the C-terminus of the VH region). MAbs with cysteines knocked into these sites are called LC_S26C, LC_S31C, HC_K64C, and HC_T120C, respectively. LC_S26C is an anti-CD3 mAb with the serine residue at position S26 of the light chain replaced by a cysteine. The other mAbs follow the same naming convention.

- 31 047669

Table 1

Sequences of the regions aHTu-CD3 VH, aHTu-CD3 VL, #2 aHTu-CD3 VH, #2 anti-IgE VL, IgG1 CH1, IgG2 CH1, IgG3 CH1, IgG4 CH1, kappa CL and lambda CL

Name of area Subsequence SEQ ID NO: Ahth-CD3 VH DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRP GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL SSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS 1 Ahth-CD3 VL DIQLTQSPATMSASPGEKVTMTCRASSSVSYMNWYQQKSGTS PKRWIYDTS KV AS G VP YRFS GS GS GTS YS LTIS S ME AED A AT Y YCQQWSSNPLTFGAGTKLELK 2 #2 anti-SVZ VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS 27 #2 anti-SPZ VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTS PKRWIYDTS KLAS G VP AHFRGS GS GTS YS LTIS GME AED A AT Y YCQQWSSNPFTFGSGTKLEIN 28 IgGl CHI ASTKGPS VFPLAPS S KSTS GGTAALGCLVKD YFPEPVTVS WNS G ALTS G VHTFP A VLQS S GLYS LS S V VT VPS S S LGTQT YICN VN HKPSNTKVDKKVEPKSC 9 IgG2 CHI ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS G ALTSGVHTFP A VLQS S GLYSLS S V VTVPS SNFGTQT YTCNVD HKPSNTKVDKTVERKCC 10 IgG3 CHI ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNS G ALTS G VHTFP A VLQS S GLYS LS SV VTVPS S S LGTQT YTCNVN HKPSNTKVDKRVELKTPLG 11 IgG4 CHI ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYG 12 Kanna CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK 13 VDNALQS GNS QES VTEQDS KDSTYSLS STLTLS KAD YEKHKV YACEVTHQGLSSPVTKSFNRGEC Lambda CL QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA DSSPVKAGVETTTTPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTEC 14

- 32 047669

Table 2

CDR regions for aHmu-CD3 mAb and #2 aHmu-CD3 mAb

Chain CDR1 SEQ ID NO: CDR2 SEQ ID NO: CDR3 SEQ ID NO: NS GYTFTRYT MN 3 YINPSRGYTNYNQK FKD 4 ARYYDDHYS LDY 5 LC RASSSVSY MN 6 DTSKVAS 7 QQWSSNPLT 8 #2 NS GYTFTRYT MN 33 YINPSRGYTNYNQK FKD 34 ARYYDDHYS LDY 35 #2 LC SASSSVSYM N 36 DTSKLAS 37 QQWSSNPFT 38

HC: heavy chain;

LC: light chain;

CDR: complementarity determining region;

CDR for anti-CDS mAb is determined using a combination of IMGT (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27:209-212) and Kabat (Elvin A. Kabat et al, Sequences of Proteins of Immunological Interest 5th ed. (1991)) methods.

Table 3

Candidate Amino Acid Substitutions for Anti-CDS mAbs for Conjugation

Region Cysteine knockin website VH CHI VL CL 1 S26C 2 K64S 3 T120C 4 S67C 5 S121C 6 Q124C 7 S119C 8 S112C 9 S25C 10 S76C 11 S27C

- 33 047669

12 S52C 13 S56C 14 K62C 15 S113C 16 K53C 17 K73C 18 Y27C 19 DIOIC 20 Q61 21 Q3 22 T5 23 S7 24 K18 25 T20 26 K45 27 Y60 28 S63 29 S65 30 S76 31 KZ 32 Q5 33 K19 34 T68 35 T70 36 S74 37 S75 38 S82a 39 Q105 40 A9 41 BY 42 S14 43 P15 44 G16 45 E17

- 34 047669

46 T42 47 S77 48 A80 49 E81 50 A100 51 K107 52 A9 53 L11 54 R13 55 A16 56 S17 57 P41 58 Q43 59 S82b 60 T83 61 S84 62 E85 63 T108 64 T110 65 D1 66 12 67 N58 68 T71 70 V54

Note: mAbs in bold were generated and shown to have significant CD3 binding activity (maximum binding was greater than 50% of the maximal antigen-binding activity of wild-type anti-CD3 mAb) after cysteine knockin. Residues where the effect of amino acid substitution on antigen-binding activity was not tested are shown in the correct form. VH and VL, Kabat numbering; CH1 and CL, EU numbering.

Four mAbs LC_S26C, LC_S31C, HC_K64C, and HC_T120C in the human HC and human kappa LC IgG4 framework were expressed in CHO cells and purified by protein A chromatography and tested for CD3 binding by FACS using Jurkat cells (Figs. 3B-3E). LC_S31C lost significant activity after cysteine knockin (Fig. 3C). LC_S26C, HC_K64C, and HC_T120C had significant CD3 binding activity (Figs. 3B and 3D, 3E) and were selected for further studies. Cysteine knockin was also performed on additional residues as shown in Table 3. 3. The resulting mAbs in the IgG4 HC and kappa LC framework were expressed in CHO cells, purified by protein A chromatography, and tested for CD3 binding by FACS using Jurkat cells. Residues after the knockin cysteine that retain more than 50% of the maximal antigen-binding activity of the wild-type anti-CD3 mAb are shown in bold in Table 3, and the results of CD3 binding by FACS using Jurkat cells are shown in Fig. 3F, 3G. Residues where the effect of amino acid substitution on antigen-binding activity was not tested are shown in the correct form (Table 3).

Example 2. Characterization of monoclonal antibodies conjugated to fatty acid molecules.

The FA molecules used for conjugation are shown in Fig. 4A, including C18, C14, C10, and C6. All molecules contain a PEG linker and a bromoacetamide-reactive group. For the conjugation reaction, the antibody was concentrated to a concentration of 20-30 mg/ml and buffer exchanged with TBS. The antibody was partially reconstituted by adding 10 equiv of tris(2-carboxyethyl)phosphine (TCEP), and the solution was incubated for 1 h at 37°C. The antibody was then buffer exchanged with DPBS and reoxidized with 30 equiv of dehydroascorbic acid by incubation for 1 h at room temperature (RT). The antibody buffer is exchanged for conjugation buffer (20 mM Tris, pH 8.5 + 150 mM NaCl + 10% glycerol) and diluted to a concentration of 10 mg/ml. The FA molecule is added in an amount of 20 equiv. from a 50 mM solution in

- 35 047669

DMSO and the resulting mixture was incubated at room temperature for 1 or 2 days. The final product was buffer exchanged with conjugation buffer to remove unreacted FA molecules. Samples were purified by hydrophobic interaction chromatography and analyzed by liquid chromatography/mass spectrometry (LC/MS). The correct conjugate on HC or LC was confirmed by mass spectrometry (MS) for each conjugated mAb (Fig. 4B-4C). Conjugation of FA to the right cysteine knockin site was confirmed by LC/MS (Table 4).

Table 4

Confirmation of FA conjugation to right cysteine knockin sites

mAb+FA m/z (z=2) (exp) m/z (z=2) (obs.) m/z (z=3) (exp.) m/z (z=3) (obs.) m/z (z=4) (exp.) m/z (z=4) (obs.) LC_S26C+C1 8 1244,99 1244,15 830,33 829,76 NSK64S+S1 8 653.34 651,86 NST120S+S 1219,05 1218,93 914,53 914,45 18 NSK64S+S6 569,14 568,77 NSK64S+S1 0 597.24 596.81 NS K64S+S1 4 625.29 624,84

Note: Conjugated mAbs are trypsin digested and analyzed by LC/MS. For a given conjugated mAb, the peak corresponding to the peptide fragment with FA conjugated to the correct cysteine knockin site is identified by LC/MS. m/z, mass-to-charge ratio, where m is the mass and z is the number of charges; exp, expected; obs, observed.

The ^8-conjugated mAbs were tested for their ability to bind to Jurkat cells (known to express CD3) in the absence or presence of 50 mg/ml bovine serum albumin (BSA) (Figs. 5A-5C). Jurkat cells were incubated with the indicated antibodies in HBSS buffer containing 0.1% casein with or without BSA during the primary antibody binding step and then treated in BSA-free buffer. Antibody binding was quantified by FACS. Binding of unconjugated mAbs to Jurkat cells was demonstrated and binding was not affected by the presence of BSA (Figs. 5A-5C). Conjugated mAbs were still able to bind to Jurkat cells (Figs. 5A-5C). Binding of conjugated mAbs to Jurkat cells was inhibited by BSA, indicating that conjugated FAs are capable of binding BSA, which subsequently reduces antigen binding of anti-CD3 mAbs. To confirm the inhibitory effect of BSA on CD3 binding by conjugated mAbs, a T cell activation assay was performed using peripheral blood mononuclear cells (PBMCs) from two different donors. PBMCs were incubated with the indicated antibodies in medium containing several concentrations of BSA for 16 h. T cell activation was assessed by measuring CD25 expression by FACS. Since the medium contains 1% FBS, a low level of BSA (approximately 0.25 mg/mL as estimated from 1% FBS) was present in each group in the assay, which was expected to suppress T cell activation by conjugated mAbs before the addition of BSA to the medium (Figs. 6A-6C). When BSA was added to the medium, increased inhibition of T cell activation by the added BSA was observed (Fig. 6A-6C).

To test the effect of the length of the conjugated FA molecule, C6, C10, and C14 FA molecules were conjugated to HC_K64C, respectively. Conjugation of each FA specifically to the heavy chain of each mAb was confirmed by LC/MS (Fig. 4C). Conjugation of FA to each cysteine knockin site was confirmed by LC/MS (Table 4). The conjugated mAbs were tested for T cell activation using PBMC from two different donors as described above (Figs. 7A-7C). C6 and C10 conjugations were less effective in blocking CD3 binding in the presence of higher concentrations of BSA (Fig. 7A-7B); C14 conjugation completely blocked CD3 binding in the presence of higher concentrations of BSA (Fig. 7C). These data indicate that longer FA molecules such as C14 and C18 are more effective in blocking CD3 binding via BSA-linked conjugation after conjugation, whereas shorter FA molecules C6 and C10 are less effective in blocking CD3 binding via BSA-linked conjugation after conjugation. Each of these characteristics may be exploited therapeutically in different tumor microenvironment conditions. For example, depending on the different albumin levels between the tumor microenvironment and circulating blood, a longer or shorter FA may be preferred as a conjugated molecule to achieve the best in vivo efficacy/safety margin.

Characterization of bispecific antibodies conjugated to fatty acid molecules.

The FA conjugation approach can be used to modulate the antigen-binding activity of one of the two arms of a bispecific antibody. For example, the bispecific antibody can be an anti-TAA/anti-CD3 T cell recruiter, wherein the FA is conjugated to the Fab region of the anti-CD3 arm (shown as the Ab1 arm in Fig. 1B, 1C). Herein, the anti-DLL3 arm is used as an example of an anti-TAA arm. The sequences used to construct the αanti-DLL3/anti-CD3 bispecific antibody (as first described in U.S. Provisional Patent Application No. 63/146,334, filed February 5, 2021, which is incorporated herein by reference in its entirety) are used to introduce cysteine knockins for FA conjugation. Cysteine is knocked into position K64 (VH region; Kabat numbering) or position T120 (CH1 region; EU numbering) of the anti-CD3 arm of the αanti-DLL3/anti-CD3 bispecific antibody. The resulting bsAbs are named bsAb HC_K64C and bsAb HC_T120C, respectively, and their sequences are listed in Table 5. Note that bsAb HC_K64C and bsAb HC_T120C share the same αanti-DLL3 arm (Table 5). The bispecific antibodies are on the backbone of human IgG1 HC and human kappa LC with the following modifications in the Fc region of IgG1: the HC anti-CD3 arm has a T366W mutation (EU numbering) to form a bulge, and the HC αanti-DLL3 arm has T366S, L368A, and Y407V mutations to form a pit. In addition, a cysteine mutation S354C is introduced in the anti-CD3 HC, and a cysteine mutation Y349C is introduced in the anti-DLL3 HC to stabilize heterodimer pairing. In addition, the mutations L234A and L235A are introduced in the CH2 regions of both H1 and H2.

- 37 047669

Table 5

Sequences of VH, VL, CH1 and CL regions of bispecific cysteine knockin antibodies for conjugation

Name of area Subsequence SEQ ID NO: bsAb NS_K64S, anti-POP VH DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKKRP GQGLEWIGYINPSRGYTNYNQKFCDKATLTTDKSSSTAYMQL S SLTSEDS A VYYC ARYYDDHYS LD YWGQGTTLT VS S 15 bsAb NS_K64S, anti-POP CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPA VLQS S GLYSLS S V VKVPS SS LGTQT YICNVN HKPSNTKVDKKVEPKSC 16 bsAb NS_K64S, anti-POP VL DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQEKSGTS PKRWIYDTS KV AS G VP YRFS GS GS GTS YS LTIS S ME AED A AT Y YCQQWSSNPLTFGAGTKLELK 17 bsAb NS_K64S, anti-POP CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKESTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 18 bsAb NS_T120S, anti-POP VH DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKKRP GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQL SSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS 19 bsAb NS_T120C, anti-POP CH1 ASCKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPA VLQS S GLYSLS SV VKVPS SS LGTQT YICNVN HKPSNTKVDKKVEPKSC 20 bsAb NS_T120S, anti-POP VL DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQEKSGTS PKRWIYDTS KV AS G VP YRFS GS GS GTS YS LTIS S ME AED A AT Y YCQQWSSNPLTFGAGTKLELK 21 bsAb NS_T120S, anti-POP CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKESTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC 22 Ahth-DLL3 EVRLSQSGGQMKKPGESMRLSCRASGYTFTSYVMHWVREAP 23

- 38 047669

VH GRRPEWIGYINPYNDATKYARKFQGRATLTSDKYSDTAFLEL RSLTSDDTAVYYCARGGYDYDGDYWGRGAPVTVSS Ahth-DLL3 CHI ASTKGPSVFPLAPSSCSTSGGTAALGCLVKDYFPEPVTVSWNS G ALTS G VHTFP A VLQS S GLYS LS S V VE VPS S S LGTQT YICN VN HKPSNTKVDKKVEPKS S 24 Ahth-DLL3 VL EIVLTQSPGTLLSLSPGERATLSCHASQNINVWLSWYQKKPGQA PRLLIYKASNLHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQGQSYPFTFGQGTKVEIK 25 Ahth-DLL3 CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQS GNS QES VTEQDS KKSTYSLS STLTLS KADYEKHKV YACEVTHQGLSSPVTKSCNRGES 26 bsAb HC_K64C, #2 anti-POP VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKKR PGQGLEWIGYINPSRGYTNYNQKFCDKATLTTDKSSSTAYMQ LS SLTSEDS A VYYC ARYYDDHYS LD YWGQGTTLTVS S 29 bsAb HC_K64C, #2 anti-POP VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQEKSGTSP KRWIYDTS KLAS G VPAHFRGS GS GTS YS LTIS GME AED A AT Y YCQQWSSNPFTFGSGTKLEIN 30 bsAb HC_T120C, #2 anti-POP VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKKR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSS 31 bsAb HC_T120C, #2 anti-POP VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQEKSGTSP KRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATY YCQQWSSNPFTFGSGTKLEIN 32

Note: VH and VL are Kabat numbering; CH1 and CL are EU numbering.

Bispecific antibodies bsAb HC_K64C and bsAb HC_T120C are transiently transfected into ExpiCHO-S cells and the simultaneous expression of two heavy chains and two light chains in the same cell results in the expression and assembly of the desired bispecific antibody and defined impurities. Impurity standards are prepared by transient transfection using the same HC and LC vectors as needed. Bispecific antibodies are first purified by Protein A chromatography. Protein A purified samples are pH adjusted to a final pH of 5.5 and loaded directly onto a poros XS (Thermo) CEX column pre-equilibrated with 25 mM phosphate (pH 5.8) + 210 mM NaCl. Samples were eluted with a linear gradient [Buffer A - 25 mM phosphate (pH 5.8) + 210 mM NaCl; Buffer B - 25 mM phosphate (pH 8) + 115 mM NaCl]. Eluted fractions were analyzed by strong cation exchange (SCE) HPLC, and fractions showing complete resolution of the 2-fold anti-OEE3 LC mismatch (HC heterodimer with anti-OEE3 LC matching on both arms) were pooled. (NH ₄ ) ₂ SO ₄ was added to the pooled fractions to a final concentration of 700 mM, and the sample was loaded directly onto a Butyl Sepharose High Performance (Cytiva) HIC (hydrophobic interaction chromatography) column pre-equilibrated with 50 mM Tris (pH 7.5) + 700 mM (NH ₄ ) ₂ SO ₄ +3% glycerol. Samples were eluted with a linear gradient [Buffer A - 50 mM Tris (pH 7.5) + 700 mM (NH ₄ ) ₂ SO ₄ +3% glycerol; Buffer B - 50 mM Tris (pH 7.5) + 10% glycerol]. Eluted fractions were analyzed by CHB-HPLC and fractions showing complete resolution of the 2-fold anti-CD3 LC mismatch (HC heterodimer with anti-CD3 LC matching on both arms) were pooled as purified protein. Purified bispecific antibodies were analyzed in three different ways.

For HPLC of CHB, samples are diluted to a final concentration of 1 mg/mL in buffer containing 1 M (NH ₄ ) ₂ SO ₄ , and 15 µL are injected directly for HPLC analysis of CHB using an Agilent column

- 39 047669

AdvanceBio HIC 4.6x100 mm 3.6 µm. (PN: 685975-908). Samples are analyzed at a flow rate of ml/min at 30°C using a linear gradient (buffer A - 50 mM Tris pH 7.5+1 M (NH ₄ )2SO ₄ ;

buffer B - 50 mM Tris pH 7.5 + 10% glycerol).

For RMSD HPLC, samples were diluted to a final concentration of 1 mg/mL in buffer containing 25 mM citrate, pH 4.5, and 15 µL were injected directly for RMSD HPLC analysis using a Waters Bioresolve SCX mAb 4.6x100 mm, 3 µm column (PN: 18609060). Samples were analyzed at a flow rate of 1 mL/min at 30°C using a linear gradient (buffer A - 25 mM phosphate, pH 5.8 + 2% ACN; buffer B - 25 mM phosphate, pH 8 + 250 mM NaCl + 2% ACN).

For size exclusion chromatography (SEC) HPLC, samples were diluted to a final concentration of 1 mg/mL in PBS, and 8 µL were injected directly for SEC HPLC analysis using an Agilent AdvanceBio SEC 300A 2.7 µm, 4.6 x 300 mm column (PN: PL1580-5301). Samples were analyzed at a flow rate of 0.35 mL/min using isocratic elution (buffer - 50 mM phosphate, pH 7.4 + 300 mM NaCl + 5% isopropanol).

The purified bsAb was analyzed by CHB-HPLC, RMSC-HPLC, and GC-HPLC (Figs. 8A, 8B, and 9A-9B). In Fig. 8A, purified bsAb HC_K64C is separated from contaminants except for the 2-fold anti-DLL3 LC mismatch by CHB-HPLC; however, when analyzed by RMSC-HPLC, purified bsAb HC_K64C is well separated from the 2-fold anti-DI .1.3 LC mismatch (Fig. 8B). These data indicate that purified bsAb HC_K64C is free of contaminants. Furthermore, purified bsAb HC_K64C is the only species by GC-HPLC (Fig. 8C). Similar observations are made with bsAb HC_T120C (Fig. 9A, 9B), indicating the high purity of the purified bsAb HC_T120C.

Purified bispecific antibodies bsAb HC_K64C and bsAb HC_T120C were conjugated to different FA molecules. For conjugation, the bispecific antibody with a cysteine knockin at position K64 or T120 was concentrated to a concentration of 20-30 mg/mL and the buffer was exchanged with TBS buffer. The bispecific antibody was partially reconstituted by adding 10 equiv. TCEP, and the solution was incubated for 1 h at 37°C. The bispecific antibody buffer was then exchanged with DPBS and re-oxidized by adding 30 equiv. dehydroascorbic acid, and the solution was incubated for 1 h at RT. The final bispecific antibody sample buffer was exchanged with conjugation buffer (20 mM Tris, pH 8.5 + 150 mM NaCl + 10% glycerol) and diluted to a concentration of 10 mg/ml. FA molecule was added in an amount of 12 equiv. from 50 mM solution in DMSO and the resulting mixture was incubated at room temperature for 1 day. The final product buffer was exchanged with conjugation buffer to remove unreacted FA molecules, and then purified by CHB purification. Purified conjugated bispecific antibodies were analyzed by CHB HPLC (Fig. 10A) and GC-HPLC (Fig. 10B). Each of the conjugated bsAbs appeared as a distinct peak with a retention time different from that of the corresponding unconjugated bsAb (Fig. 10A), indicating high conjugation efficiency. In addition, each conjugated bsAb appeared as a distinct peak on the HPLC-SEC (Fig. 10B).

Table 6

Confirmation of FA conjugation with bsAbs

bsAb Calculated mm Observed mm Notes Parent bsAb 145307,4 145310,7 Expected bsAb НС К64С 145282,4 145404,8 + 1 cysteine bsAb НС Т120С 145309,4 145433,0 + 1 cysteine bsAbHCK64CC10 145963,3 145967,4 Expected bsAbHCK64CC14 146019,4 146022,5 Expected bsAbHCK64CC18 146075,5 146079,1 Expected bsAbHC T120C C14 146046,4 146051,2 Expected bsAbHC T120C C18 146102,5 146106,6 Expected

Note: Parent bsAb, anti-DLL3/anti-CD3 bsAb without knocked-in cysteine; mm, molecular weight; + 1 cysteine, one cysteine is expected to be covalently linked to the knocked-in cysteine, and the obtained mm is as expected.

To assess the binding activity of unconjugated and conjugated bispecific antibodies to both DLL3 and CD3, purified bispecific antibodies were incubated with SHP77 cells and Jurkat cells labeled with different fluorescent markers. Double staining events induced by the bispecific antibody were detected and quantified by flow cytometry. Briefly, Jurkat cells were stained with Violet Proliferation Dye 450 (BD, Cat: 562158) and SHP-77 cells were stained with CFSE (ThermoFisher, Cat: 34554) according to

- 40 047669 with the manufacturer's protocol. Then, labeled SHP-77 and Jurkat cells at a 1:1 ratio were incubated with 2 μg/ml bsAb in the presence or absence of 1.5 μM blocking anti-DLL3 mAb or 1.5 μM blocking anti-CD3 mAb (Fig. 11). When using blocking mAbs, SHP-77 cells were pretreated with 4.5 μM blocking anti-DLL3 mAb for 10 min at room temperature prior to incubation with Jurkat cells at a final concentration of 1.5 μM blocking anti-DLL3 mAb, or Jurkat cells were pretreated with 4.5 μM blocking anti-CD3 mAb for 10 min at room temperature prior to incubation with SHP-77 cells at a final concentration of 1.5 μM blocking anti-CD3 mAb. After incubation in a CO ₂ incubator at 37°C for 1 h, cells were fixed with 2% formaldehyde, washed once with TBS, resuspended in FACS buffer (HBSS, 0.1% BSA, 0.05% sodium azide), and then analyzed by flow cytometry (Attune NxT). Cross-linking of the two cell types in the presence of bsAb was detected by FACS, and the signal for each bsAb (unconjugated and conjugated) was inhibited by blocking anti-DLL3 or anti-CD3 mAb (Fig. 11). These data demonstrate that unconjugated and conjugated bsAbs can bind to two different antigens simultaneously.

Bispecific antibodies are also used to activate T cells in a functional T cell activation assay. The Jurkat NFAT luciferase reporter cell line (BPS Bioscience) is used, which conditionally expresses firefly luciferase upon activation (including CD3-mediated activation). Reporter cells are incubated with SHP-77 target cells in the presence of each bsAb (unconjugated and conjugated) and with or without anti-DLL3 blocking antibody (at a final concentration of 500 nM) for 22 h in growth medium at 37°C in a CO ₂ incubator. Cells are then assayed for activation using luciferase detection reagent and a luminometer. Each of the bispecific antibodies induced dose-dependent activation of reporter cells when incubated with the SHP-77 target cell, and the signal was inhibited by a blocking anti-DLL3 antibody (an anti-DLL3 arm version of the mAb) (Fig. 12A-12B), demonstrating the T cell-attracting function of the bispecific antibodies. Since 0.5% FBS (fetal bovine serum) is required as part of the culture medium for cell survival during the assay, a low level of BSA (approximately 0.13 mg/mL as estimated from 0.5% FBS) is present in the assay, which is expected to result in a decrease in the T cell activation signal by bsAb HC_K64C_C10, bsAb HC_K64C_C14, and bsAb HC_K64C_C18 (Fig. 12A).

The T cell activation assay using the Jurkat NFAT luciferase reporter cell line was also performed in the presence of low and high levels of BSA to assess the inhibitory effect of albumin on the T cell activation function of the conjugated bispecific antibodies. Since 0.5% FBS is required as part of the culture medium for cell survival during the assay, a low level of BSA (approximately 0.13 mg/mL as estimated from 0.5% FBS) was present in each assay group. When a high level of BSA (final concentration of 10 mg/mL BSA in addition to 0.5% FBS) was added to the assay, the T cell activation induced by the conjugated bispecific bsAb HC_K64C_C14 and bsAb HC_K64C_C18 was inhibited compared to the control group (0.5% FBS only) (Fig. 13A); High level of BSA (10 mg/ml BSA+0.5% FBS) inhibited T cell activation induced by conjugated bispecific bsAbs HC_T120C_C14 and bsAb HC_T120C_C18 compared to the control group (0.5% FBS only) (Fig. 13B). These data demonstrate that the activity of anti-DLL3/anti-CD3 bispecific antibodies conjugated to FA molecules can be modulated by BSA levels. Fig. 5B shows that conjugation of FA to mAb HC_K64C does not alter the binding of the conjugated arm to CD3; further, Fig. 11 shows that FA conjugation to bsAb HC_K64C does not result in a dramatic change in its bispecific activity, suggesting that the lower activity of bsAb HC_K64C_C10 in Fig. 13A is a consequence of the low level of BSA tolerated by the 0.5% FBS required as part of the culture medium. Similar observations are made for HC_K64C_C14 and HC_K64C_C18 (Fig. 13A). These observations are consistent with the data in Figs. 6B and 7A-7C, which show that FA conjugation at position K64 affects binding of the anti-CD3 arm to CD3 much more than at position T120 in the presence of BSA. This is also consistent with the fact that BSA bound to FA conjugated at K64C is closer to the CDR and can block target antigen (CD3) binding more efficiently than at T120C.

An ELISA assay was performed to evaluate the effect of BSA on the antigen-binding activity of the anti-DLL3 arm of each conjugated bispecific antibody. A 96-well ELISA plate was coated with DLL3 protein (Adipogen, Cat#: AG-40B-0151-C010) for 1 h at room temperature, followed by blocking with 5% BSA in TBST for 1 h at RT. The plate was washed 3 times with TBST and pre-incubated at room temperature for 1 h with or without blockers (200 μg/ml anti-DLL3 F(ab') ₂ or 50 mg/ml BSA (Sigma, Cat#: A4612-25G); TBST was used for the no-blocker groups). The plate was then incubated with 1 μg/ml bsAb for 30 min at RT in the presence or absence of 100 μg/ml anti-DLL3 F(ab') ₂ or 50 mg/ml BSA. Following incubation, the plate was washed and the signal was detected with HRP-conjugated anti-human IgG secondary antibody (ThermoFisher, Cat# H10007) and TMB substrate (ThermoFisher, Cat# 34029) using an Envision spectrophotometer. Figure 14 shows that BSA had very little effect on the antigen-binding activity of the anti-DLL3 arm of each conjugated bsAb.

A second set of anti-CD3 VH and VL sequences (#2 anti-CD3) (SEQ ID NOs:27 and 28, respectively; Kabat numbering) can also be used to construct conjugated mAbs and bsAbs and are listed in Table 1. In addition, modified versions of these sequences can also be used to construct conjugated anti-DLL3/anti-CD3 bispecific antibodies (Table 5) comprising a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:29, a VL region with the polypeptide sequence of SEQ ID NO:30, a CH1 region with the polypeptide sequence of SEQ ID NO:16, and a CL region with the polypeptide sequence of SEQ ID NO:18; or a first antigen-binding arm (Ab1) comprising a VH region with a polypeptide sequence of SEQ ID NO:31, a VL region with a polypeptide sequence of SEQ ID NO:32, a CH1 region with a polypeptide sequence of SEQ ID NO:20, and a CL region with a polypeptide sequence of SEQ ID NO:22. In each of the above cases, the second antigen-binding arm (Ab2) comprises a VH region with a polypeptide sequence of SEQ ID NO:23, a VL region with a polypeptide sequence of SEQ ID NO:25, a CH1 region with a polypeptide sequence of SEQ ID NO:24, and a CL region with a polypeptide sequence of SEQ ID NO:26. In addition, each of the above-mentioned first antigen-binding arms (Ab1) can be used to construct anti-CD3 bispecific antibodies and TAAs other than DLL3.

It will be understood by those skilled in the art that changes can be made to the embodiments described above without departing from their broad inventive concept. It should therefore be understood that this invention is not limited to the specific embodiments described, but is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

Claims (28)

CLAUSE OF THE INVENTION 1) a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:19, a VL region with the polypeptide sequence of SEQ ID NO:21, a CH1 region with the polypeptide sequence of SEQ ID NO:20 and a CL region with the polypeptide sequence of SEQ ID NO:22; 1) a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12, with an amino acid substitution T120C, as defined by EU numbering, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14; (1) residue 25, 27, 62, 64, 73, 76, 101, 112 or 113 of SEQ ID NO:1 or 27 as determined by Kabat numbering; (1) the K64C substitution in SEQ ID NO:1, as defined by Kabat numbering; (1) residue 25, 27, 62, 64, 73, 76, 101, 112 or 113 of SEQ ID NO:1, as determined by Kabat numbering; 1. An isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: a) variable region of the heavy chain (VH); b) variable region of the light chain (VL); where the antibody or its antigen-binding fragment binds to the target antigen; an amino acid residue in VH, VL, or within five (5) amino acids of VH or VL on one or both arms is replaced by an amino acid residue that is conjugated to a fatty acid (FA); when conjugated to FA at the substituted amino acid residue, the monoclonal antibody or antigen-binding fragment thereof still binds to the target antigen; and FA conjugated to an antibody or its antigen-binding fragment is capable of binding albumin, where the binding of albumin to FA results in: (a) partial or complete blocking of target antigen binding to the antibody or antigen-binding fragment thereof; and/or (b) reduced ability to activate T cells when bound to albumin compared to an isolated antibody or antigen-binding fragment thereof not bound to albumin; where the replaced amino acid residue is a cysteine residue or a lysine residue; FA is selected from FA with 6 carbon atoms, 8 carbon atoms, 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 16 carbon atoms or 18 carbon atoms or any number of carbon atoms therebetween. 2) a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:15, a VL region with the polypeptide sequence of SEQ ID NO:17, a CH1 region with the polypeptide sequence of SEQ ID NO:16 and a CL region with the polypeptide sequence of SEQ ID NO:18; 2) a VH region with the polypeptide sequence of SEQ ID NO:1 with the amino acid substitution K64C, as determined by Kabat numbering, and a VL region with the polypeptide sequence of SEQ ID NO:2; (2) residue 26, 27, 52, 53, 56 or 67 of SEQ ID NO:2 or 28, as determined by Kabat numbering; (2) a substitution of S26C in the sequence of SEQ ID NO:2, as defined by Kabat numbering, or (3) a substitution of T120C in the sequence of SEQ ID NO:9, 10, 11 or 12, as defined by EU numbering. - 42 047669 (2) residue 26, 27, 52, 53, 56 or 67 of SEQ ID NO:2, as determined by Kabat numbering; 2. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the replaced amino acid residue is at an amino acid residue corresponding to: 3) a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:29, a VL region with the polypeptide sequence of SEQ ID NO:30, a CH1 region with the polypeptide sequence of SEQ ID NO:16 and a CL region with the polypeptide sequence of SEQ ID NO:18; or 3) a VH region with a polypeptide sequence of SEQ ID NO:27 with an amino acid substitution of K64C, as determined by Kabat numbering, and a VL region with a polypeptide sequence of SEQ ID NO:28; (3) residue 119 or 120 of SEQ ID NO:9, 10, 11 or 12, as defined by EU numbering; or (4) residue 121 or 124 of SEQ ID NO:13 or 14, as defined by EU numbering. 3. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the replaced amino acid residue is at an amino acid residue corresponding to: (3) residue 119 or 120 of SEQ ID NO:9, 10, 11 or 12, as defined by EU numbering or (4) residue 121 or 124 of SEQ ID NO:13 or 14, as defined by EU numbering. 4) a first antigen-binding arm (Ab1) comprising a VH region with the polypeptide sequence of SEQ ID NO:31, a VL region with the polypeptide sequence of SEQ ID NO:32, a CH1 region with the polypeptide sequence of SEQ ID NO:20 and a CL region with the polypeptide sequence of SEQ ID NO:22. 4) a VH region with the polypeptide sequence of SEQ ID NO:1 and a VL region with the polypeptide sequence of SEQ ID NO:2 with the amino acid substitution S26C, as determined by Kabat numbering; 4. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is an anti-immune cell modulator (ICM) antibody or antigen-binding fragment thereof and is capable of specifically binding to ICM, optionally wherein ICM is selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 4-1BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules. 5) a VH region with the polypeptide sequence of SEQ ID NO:27, and a VL region with the polypeptide sequence of SEQ ID NO:28 with the amino acid substitution S26C, as determined by Kabat numbering; 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the ICM is CD3 and the antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, with the polypeptide sequences of SEQ ID NOs: 3, 4, 5, 6, 7 and 8, respectively; or SEQ ID NOs: 33, 34, 35, 36, 37 and 38, respectively. 6) a VH region with a polypeptide sequence of SEQ ID NO:1, a VL region with a polypeptide sequence of SEQ ID NO:2, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12 with an amino acid substitution T120C, as defined by EU numbering, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14; or 6. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the replaced amino acid residue is at an amino acid residue selected from: 7) a VH region with a polypeptide sequence of SEQ ID NO:27, a VL region with a polypeptide sequence of SEQ ID NO:28, a CH1 region with a polypeptide sequence selected from SEQ ID NO:9, 10, 11 or 12 with an amino acid substitution T120C, as defined by EU numbering, and a CL region with a polypeptide sequence selected from SEQ ID NO:13 or 14. 7. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 4-6, comprising: 8. An isolated multispecific antibody or antigen-binding fragment thereof comprising a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the multispecific antibody or antigen-binding fragment thereof comprises one or more antigen-binding arms comprising a substituted amino acid residue that is conjugated to FA. 9. A multispecific antibody or antigen-binding fragment thereof according to claim 8, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof comprising a first antigen-binding arm (Ab1) and a second antigen-binding arm (Ab2), wherein Ab1 and/or Ab2 comprise a substituted amino acid that is conjugated to FA. 10, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, wherein Ab2 binds a tumor-associated antigen (TAA), optionally wherein the TAA is DLL3. 10. The isolated multispecific antibody or antigen-binding fragment thereof according to claim 9, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, wherein Ab1 binds an immune cell modulator (ICM), optionally wherein the ICM is selected from the group consisting of CD3, CD27, CD28, CD40, CD122, OX40, CD16, 41BB, GITR, ICOS, CTLA-4, PD-1, LAG-3, TIM-3, TIGIT, VISTA, SIGLEC7, NKG2D, SIGLEC9, KIR, CD91, BTLA, NKp46, B7-H3, SIPRa and other cell surface immunoregulatory molecules. - 43 047669 11. An isolated multispecific antibody or antigen-binding fragment thereof according to claim 9 or 12. An isolated multispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 11, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof comprising: 13. An isolated multispecific antibody or antigen-binding fragment thereof according to claim 12, wherein the multispecific antibody or antigen-binding fragment thereof is a bispecific antibody or antigen-binding fragment thereof, wherein the second antigen-binding arm (Ab2) comprises a VH region with the polypeptide sequence of SEQ ID NO:23, a VL region with the polypeptide sequence of SEQ ID NO:25, a CH1 region with the polypeptide sequence of SEQ ID NO:24, and a CL region with the polypeptide sequence of SEQ ID NO:26. 14. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or an isolated multispecific antibody or antigen-binding fragment thereof according to any one of claims 8 to 13, wherein the FA comprises a linker for conjugation to a substituted amino acid residue, wherein the linker is selected from a peptide linker or a polyethyleneglycol linker, optionally wherein the peptide linker consists of less than 50 amino acids. 15. An isolated nucleic acid encoding an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 14. 16. An isolated nucleic acid encoding an isolated multispecific antibody or antigen-binding fragment thereof according to any one of claims 8 to 14. 17. An expression vector containing the isolated nucleic acid according to claim 15 or 16. 18. An isolated host cell containing the vector according to claim 17. 19. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7 and 14 or an isolated multispecific antibody or antigen-binding fragment thereof according to any one of claims 8-14 and a pharmaceutically acceptable carrier. 20. A method for treating cancer in a patient, wherein the method comprises administering to the patient a pharmaceutical composition according to claim 19. 21. The method according to claim 20, wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, and non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other hematological malignancies. 22. A method for obtaining an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1-7 and 14, wherein the method comprises: culturing a cell according to paragraph 18 under conditions of producing an antibody or its antigen-binding fragment; isolating the antibody or its antigen-binding fragment from the cell or culture and conjugating FA to the antibody or its antigen-binding fragment at the replaced amino acid residue. - 44 047669 23. A method for producing an isolated multispecific antibody or antigen-binding fragment thereof according to any one of claims 8-14, wherein the method comprises culturing a cell according to claim 18 under conditions for producing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof from the cell or culture and conjugating FA to the antibody or antigen-binding fragment thereof at the replaced amino acid residue. 24. A method for producing a pharmaceutical composition containing an isolated antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein the method comprises combining the antibody or antigen-binding fragment thereof according to any one of claims 1-14 with a pharmaceutically acceptable carrier to produce the pharmaceutical composition. 25. A method for partially or completely blocking the binding between a target antigen and an antibody or antigen-binding fragment, comprising contacting albumin with an isolated antibody or its antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or its antigen-binding fragment is capable of specifically binding to the target antigen, FA is capable of binding to albumin, and the binding of albumin to FA results in a partial or complete block of the binding of the target antigen to the antibody or its antigen-binding fragment. 26. The method according to claim 25, wherein the step of bringing into contact comprises administering a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof to a patient in need of treatment of a tumor, wherein the tumor contains the target antigen. 27. The method according to claim 25 or 26, wherein the albumin has a higher metabolism in the tumor microenvironment compared to circulating blood and/or is present in the tumor microenvironment at a level lower than the level of albumin in the circulating blood of the subject. 28. The method according to claim 27, wherein the lower level of albumin in the tumor microenvironment is due to high albumin catabolism in the tumor microenvironment and/or high levels of proteases in the tumor microenvironment.