HK40112932A - Anti-5t4 antibodies and uses thereof - Google Patents

Description

Anti-5T4 antibodies and their uses

Background Technology

5T4 (also known as trophoblast glycoprotein, TPBG; 5T4 carcinoembryonic trophoblast glycoprotein; and Wnt activation inhibitor 1, WAIF1) is a vertebrate-specific, single-pass transmembrane protein, first identified in human placental tissue. 5T4 contains a highly glycosylated rigid core, including eight leucine-rich repeat sequences (LRRs) in its extracellular domain, a transmembrane helix, and a cytoplasmic region. The cytoplasmic PDZ-binding motif Ser-Asp-Val of 5T4 has been reported to interact with the PDZ domain of TIP-2/GIPC, a cytoplasmic protein that associates with vesicles located near the cell membrane. Other downstream mechanisms of signal transduction remain unknown. 5T4 was also found to inhibit the Wnt/β-catenin signaling pathway, a key pathway in embryonic development and a major target for anticancer therapy.

5T4 is rarely expressed in normal adult tissues, but it is present at high levels in the placenta and most common tumors, typically accounting for more than 80% in kidney cancer, breast cancer, colon cancer, prostate cancer, and ovarian cancer. Therefore, 5T4 is characterized by carcinoembryonic antigen, highlighting its potential as a diagnostic biomarker or target for cancer treatment.

Summary of the Invention

In several embodiments, this disclosure provides antibodies and antigen-binding fragments specific to the human 5T4 protein. Experimental tests have shown that these newly identified antibodies bind to the human 5T4 protein efficiently and specifically. In contrast to natropumab, a fusion protein containing a Fab fragment targeting 5T4 and which has been evaluated in clinical trials, these newly identified antibodies also bind to the cynomolgus monkey 5T4 protein with comparable potency.

According to one embodiment of this disclosure, an antibody or antigen-binding fragment thereof is provided that is specific for human 5T4 carcinoembryonic trophoblast glycoprotein (5T4) protein and includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region including VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region including VL CDR1, VL CDR2 and VL CDR3.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 41, 42 (or any one of 47-53), 43, 44, 45, and 46 (or 264 or 265), respectively. In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO: 41, VH CDR2 contains the amino acid sequence of SEQ ID NO: 42, VH CDR3 contains the amino acid sequence of SEQ ID NO: 43, VL CDR1 contains the amino acid sequence of SEQ ID NO: 44, VL CDR2 contains the amino acid sequence of SEQ ID NO: 45, and VL CDR3 contains the amino acid sequence of SEQ ID NO: 264 or 265. In some embodiments, VH contains the amino acid sequence of SEQ ID NO:197, and VL contains the amino acid sequence of SEQ ID NO:262 or 263.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 54, 55 (or any one of 60-63 or 266 or 267), 56, 57, 58, and 59. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 64-69. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 70, 71, or 76, 72, 73, 74, or 75. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 each contain the amino acid sequence of SEQ ID NO:77-82.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 83-88. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 89-94. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 95, 96 (or any one of 101-104), 97, 98, 99, and 100. In some embodiments, VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 105-110. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 111-116.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 117-122. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 123, 124 or 129, 125, 126, 127, and 128. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 130-135. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 136-141. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 142-147.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 148-153. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 154-159. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 160, 161 or 166, 162, 163, 164, and 165. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 167, 168, or 173, 169, 170, 171, and 172, respectively. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 174, 175, or 180, 176, 177, 178, and 179, respectively.

Antibody-drug conjugates comprising an antibody or a fragment thereof conjugated with a pharmaceutical moiety of the present disclosure are also provided. In some embodiments, the pharmaceutical moiety is a cytotoxic agent or a cell inhibitor. In some embodiments, the pharmaceutical moiety is maytansinoid, auristatin, or a macrolide analog. In some embodiments, the pharmaceutical moiety comprises monomethylauuristatin E (MMAE) or monomethylauuristatin F (MMAF). In some embodiments, the pharmaceutical moiety is linked to an antibody or a fragment thereof via a hydrolyzable linker under acidic conditions.

In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO:41, VH CDR2 contains the amino acid sequence of SEQ ID NO:42, VH CDR3 contains the amino acid sequence of SEQ ID NO:43, VL CDR1 contains the amino acid sequence of SEQ ID NO:44, VL CDR2 contains the amino acid sequence of SEQ ID NO:45, and VL CDR3 contains the amino acid sequence of SEQ ID NO:264 or 265. In some embodiments, VH contains the amino acid sequence of SEQ ID NO:197, and VL contains the amino acid sequence of SEQ ID NO:262 or 263.

Multispecific antibodies are also provided, which comprise the antigen-binding fragment of this disclosure and one or more antibodies or antigen-binding fragments having binding specificity to non-5T4 target antigens.

In another embodiment, a chimeric antigen receptor (CAR) is also provided, which includes the antigen-binding fragment, transmembrane domain, co-stimulatory domain, and CD3ξ intracellular domain of the present disclosure.

Polynucleotides are also provided that encode the disclosed antibodies or their antigen-binding fragments or CARs. In some embodiments, the polynucleotides are optionally chemically modified mRNA.

Methods and uses for treating cancer and inflammatory conditions using the antibodies or antigen-binding fragments of the present disclosure are also provided.

Attached Figure Description

Figure 1 shows the ELISA binding activity of the tested anti-5T4 chimeric monospecific antibody to human 5T4 protein.

Figure 2 shows the ELISA binding activity of the tested anti-5T4 chimeric monospecific antibody to cynomolgus monkey 5T4 protein.

Figures 3 through 8 show the epitope binning of the anti-5T4 chimeric monospecific antibody as tested by competitive ELISA assay.

Figure 9 shows the binding activity of the tested anti-5T4 chimeric mAb to human 5T4 expressed on the surface of CHO-K1.

Figure 10 shows that most humanized 14G12 antibodies have similar binding activity to human 5T4 antigen compared to chimeric 14G12 mAbs.

Figure 11 shows that most of the tested humanized 14G12 antibodies had similar binding activity to CHOK1 overexpressing human 5T4 compared to chimeric 14G12 antibodies.

Figure 12 shows that the tested humanized 393E9 antibody and its chimeric mAb have binding activity comparable to that of the human 5T4 antigen.

Figure 13 shows that some tested humanized 393E9 antibodies have similar or even stronger binding activity to chimeric antibodies on CHOK1-hu5T4 cells.

Figure 14 shows that the tested humanized 159D5 antibody and its chimeric mAb have binding activity comparable to that of the human 5T4 antigen.

Figure 15 shows that the tested humanized 159D5 antibodies and their chimeric antibodies have considerable binding activity to CHOK1 overexpressing human 5T4.

Figure 16 shows that some tested humanized 286B4 antibodies exhibited similar binding activity to the human 5T4 antigen compared to chimeric antibodies.

Figure 17 shows that some of the tested humanized 286B4 antibodies have similar or even stronger binding activity to their chimeric antibodies on CHOK1-hu5T4 or MCF-7 cells.

Figure 18 shows that antibodies that remove PTM have enhanced binding ability to cells expressing 5T4 compared to their chimeric counterparts.

Figure 19 shows that the affinity-matured antibodies have stronger binding activity to the human 5T4 antigen than their parent Hu14G12-28 antibody.

Figure 20 shows that the affinity-matured antibodies have enhanced binding ability to cells expressing 5T4 compared to their parent Hu14G12-28 antibody.

Detailed Implementation

definition

It should be noted that the term "a" or "an" entity refers to one or more entities within that entity; for example, "an antibody" should be understood to mean one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this document.

As used herein, an "antibody" or "antigen-binding" polypeptide refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment thereof, or a single chain. Therefore, the term "antibody" includes any protein or peptide containing a molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Examples include, but are not limited to, the complementarity-determining region (CDR) or ligand-binding portion of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region, or any portion thereof, or at least a portion of the binding protein.

As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror iron, and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen to form a complex.

The term antibody encompasses major categories of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as γ, μ, α, δ, or ε, and some subclasses thereof (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE.

Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to have functional specializations. Given this disclosure, the modifications of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore within the scope of this disclosure. All immunoglobulin classes are obviously within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000–70,000 Daltons. These four chains are typically linked by disulfide bonds in a “Y” configuration, where the light chain surrounds the heavy chain, starting at the opening of the “Y” and continuing through the variable region.

The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primate-derived antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs(scFv)), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

As used herein, the term "chimeric antibody" will be understood to refer to any antibody in which the immune-reactive region or site is derived from or originates from a first species and the constant region (which, according to this disclosure, may be whole, partial, or modified) is derived from a second species. In some embodiments, the target-binding region or site will be derived from a non-human source (e.g., mouse or primate), and the constant region will be human.

The antibodies disclosed herein can be derived from any animal source, including birds and mammals. Preferably, the antibodies are from humans, mice, donkeys, rabbits, goats, guinea pigs, camels, llamas, horses, or chickens. In some embodiments, the variable region may be of condricthoid origin (e.g., from sharks).

As used herein, the term “recombination” when referring to a polypeptide or polynucleotide means a form of polypeptide or polynucleotide that does not exist naturally, and non-limiting examples of which can be produced by combining polynucleotides that do not normally appear together.

Hybridoma technology can be performed under conditions of varying “toughness.” Typically, low-toughness hybridization reactions are carried out at approximately 40°C in solutions of approximately 10 × SSC or equivalent ionic strength/temperature. Medium-toughness hybridization is typically carried out at approximately 50°C in solutions of approximately 6 × SSC, and high-toughness hybridization reactions are typically carried out at approximately 60°C in solutions of approximately 1 × SSC. Hybridization reactions can also be performed under “physiological conditions” well known to those skilled in the art. Non-limiting examples of physiological conditions include temperature, ionic strength, pH, and ^Mg²⁺ concentration typically present in cells.

Anti-5T4 antibody

As shown in the accompanying experimental examples, the inventors were able to generate anti-5T4 antibodies 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10 (Table 1). Equally important, many of these antibodies exhibited higher biological activity than naptumab (NeoTX), a benchmark fusion protein containing the Fab moiety targeting 5T4. Furthermore, these antibodies showed cross-reactivity with cynomolgus monkey 5T4, which allowed for further preclinical evaluation.

Competitive binding assays revealed that these newly identified antibodies, along with napotumab, could be classified into four distinct bins based on their binding sites on the 5T4 antigen. Interestingly, only 14G12, 393E9, and 113H5 competed with napotumab for binding to 5T4 (referred to as "Bin A," Table 4). Binding bins included 159D5, 24F10, and 493E10; bin C included 257F1, 353H11, 367B8, 389G2, and 109H7; and bin D included 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10. Sequence analysis showed that certain CDRs of the antibodies within each of these bins were highly homologous and therefore considered interchangeable.

According to one embodiment of this disclosure, an antibody or an antigen-binding fragment thereof is provided. In some embodiments, the antibody or the antigen-binding fragment thereof has binding specificity to human 5T4 protein. In some embodiments, the antibody or the antigen-binding fragment thereof includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region including VH CDR1, VH CDR2, and VHCDR3, and the light chain variable region including VL CDR1, VL CDR2, and VL CDR3.

Sequence analysis revealed that some CDRs may be post-translational modified. To avoid the risk of post-translational modification (PTM) and thus simplify manufacturing, this disclosure designs and tests certain risk-free forms of CDRs.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 393E9 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:54; VH CDR2 comprises the amino acid sequence of SEQ ID NO:55; VH CDR3 comprises the amino acid sequence of SEQ ID NO:56; VL CDR1 comprises the amino acid sequence of SEQ ID NO:57; VLCDR2 comprises the amino acid sequence of SEQ ID NO:58; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:59.

In some embodiments, VH CDR2 is derisked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:54; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:55 or SEQ ID NO:60-63 or 266-267; VH CDR3 comprises the amino acid sequence of SEQ ID NO:56; VL CDR1 comprises the amino acid sequence of SEQ ID NO:57; VL CDR2 comprises the amino acid sequence of SEQ ID NO:58; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:59.

In some embodiments, VH CDR2 is de-risked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 54; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 266; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 58; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO: 59. In some embodiments, VHCDR2 is de-risked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 54; VHCDR2 comprises the amino acid sequence of SEQ ID NO: 267; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 56; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 57; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 58; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO: 59. Interestingly, as shown in Example 12, the derisked forms of PTM, particularly Hu393E9-45-P2 and Hu393E9-62-P2 (both containing SEQ ID NO: 267 as VH CDR2), exhibit enhanced affinity compared to the chimeric forms.

The exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:3, 215-221, and 248-256. The exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:4 and 222-226.

In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:3, 215-221, and 248-256, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:3, 215-221, and 248-256, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:4 and 222-226, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:4 and 222-226, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 393E9 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 393E9 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 286B4 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:123; VH CDR2 comprises the amino acid sequence of SEQ ID NO:124; VH CDR3 comprises the amino acid sequence of SEQ ID NO:125; VL CDR1 comprises the amino acid sequence of SEQ ID NO:126; VL CDR2 comprises the amino acid sequence of SEQ ID NO:127; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:128.

In some embodiments, VH CDR2 is derisked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:123; VH CDR2 comprises the amino acid sequence of SEQ ID NO:124 or SEQ ID NO:129; VHCDR3 comprises the amino acid sequence of SEQ ID NO:125; VL CDR1 comprises the amino acid sequence of SEQ ID NO:126; VLCDR2 comprises the amino acid sequence of SEQ ID NO:127; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:128. Interestingly, as shown in Example 12, the PTM-derisked form exhibits enhanced affinity compared to the chimeric form.

The exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:23, 236-241, and 259-261. The exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:24 and 242-247.

In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:23, 236-241, and 259-261, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:23, 236-241, and 259-261, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:24 and 242-247, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:24 and 242-247, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 286B4 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 286B4 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 14G12 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of SEQ ID NO:42; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VLCDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46.

In some embodiments, VH CDR2 is derisked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46.

In some implementations, VL CDR3 is affinity matured. As shown in Example 20, the affinity matured antibodies Hu14G12-28-88# and Hu14G12-28-108# showed significantly increased binding affinity to human 5T4 protein by 9.45-fold and 7.41-fold, respectively, compared to the parental 14G12-28 antibody.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of SEQ ID NO:42; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:264. In some embodiments, VH CDR2 is de-risked by PTM. In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO:41; VH CDR2 contains the amino acid sequence of SEQ ID NO:42; VHCDR3 contains the amino acid sequence of SEQ ID NO:43; VL CDR1 contains the amino acid sequence of SEQ ID NO:44; VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and VL CDR3 contains an amino acid sequence selected from SEQ ID NO:265.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46, 264, and 265.

Exemplary VH sequences include amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. Exemplary VL sequences include amino acid sequences selected from SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:263. Yet another exemplary VH sequence includes an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:263.

In some embodiments, VH includes the amino acid sequence of any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, while retaining the corresponding VL CDR or its PTM de-risked form. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:197, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:262, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:262, while retaining the corresponding VL CDR or its affinity mature form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:263, while retaining the corresponding VL CDR or its affinity mature form.

In some embodiments, an antibody-antigen binding fragment that binds to the same epitope on 5T4 as 14G12 is also provided. In some embodiments, an antibody-antigen binding fragment that competes with 14G12 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 159D5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:70; VH CDR2 comprises the amino acid sequence of SEQ ID NO:71; VH CDR3 comprises the amino acid sequence of SEQ ID NO:72; VL CDR1 comprises the amino acid sequence of SEQ ID NO:73; VLCDR2 comprises the amino acid sequence of SEQ ID NO:74; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:75.

In some embodiments, VH CDR2 is derisked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:70; VH CDR2 comprises the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:76; VHCDR3 comprises the amino acid sequence of SEQ ID NO:72; VL CDR1 comprises the amino acid sequence of SEQ ID NO:73; VL CDR2 comprises the amino acid sequence of SEQ ID NO:74; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:75. As shown in Example 12, the PTM-derisked form (159D50-P1) has similar properties to the chimeric antibody.

The exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:7, 227-229, and 257. The exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:8, 230-235, and 258.

In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:7, 227-229, and 257, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:7, 227-229, and 257, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:8, 230-235, and 258, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:8, 230-235, and 258, while retaining the corresponding VLCDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 159D5 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 159D5 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 353H11 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:95; VH CDR2 comprises the amino acid sequence of SEQ ID NO:96; VH CDR3 comprises the amino acid sequence of SEQ ID NO:97; VL CDR1 comprises the amino acid sequence of SEQ ID NO:98; VL CDR2 comprises the amino acid sequence of SEQ ID NO:99; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:100.

In some embodiments, VH CDR2 is derisked by PTM. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:95; VH CDR2 comprises the amino acid sequence of either SEQ ID NO:96 or SEQ ID NO:101-104; VH CDR3 comprises the amino acid sequence of SEQ ID NO:97; VL CDR1 comprises the amino acid sequence of SEQ ID NO:98; VL CDR2 comprises the amino acid sequence of SEQ ID NO:99; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:100.

The exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:15, and the exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:16. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:15, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:15, while retaining the corresponding VHCDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:16, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:16, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 353H11 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 353H11 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 109H7 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:117; VH CDR2 comprises the amino acid sequence of SEQ ID NO:118; VH CDR3 comprises the amino acid sequence of SEQ ID NO:119; VL CDR1 comprises the amino acid sequence of SEQ ID NO:120; VL CDR2 comprises the amino acid sequence of SEQ ID NO:121; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:122.

The exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:21, and the exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:22. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:21, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:21, while retaining the corresponding VHCDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:22, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 109H7 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 109H7 for binding to 5T4 is therefore also provided.

In some embodiments, an antibody or antigen-binding fragment derived from antibody 49C5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:154; VH CDR2 comprises the amino acid sequence of SEQ ID NO:155; VH CDR3 comprises the amino acid sequence of SEQ ID NO:156; VL CDR1 comprises the amino acid sequence of SEQ ID NO:157; VL CDR2 comprises the amino acid sequence of SEQ ID NO:158; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:159.

The exemplary VH sequence comprises the amino acid sequence of SEQ ID NO:33, and the exemplary VL sequence comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:33, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:33, while retaining the corresponding VHCDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:34, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:34, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, an antibody and antigen-binding fragment that binds to the same epitope on 5T4 as 49C5 is also provided. In some embodiments, an antibody and antigen-binding fragment that competes with 49C5 for binding to 5T4 is therefore also provided.

Antibodies comprising a group of CDRs (VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3) including any of the antibodies disclosed herein, such as 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10 (Table 1) or their PTM de-risked forms, are also provided. CDR sequences are described in Tables 1-1 to 1-41.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 41, 42 (or any one of 47-53), 43, 44, 45, and 46 (or 264 or 265). In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 54, 55 (or any one of 60-63 or 266 or 267), 56, 57, 58, and 59. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 64-69. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VLCDR3 each comprise the amino acid sequences of SEQ ID NO: 70, 71, 76, 72, 73, 74, or 75. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 77-82.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 83-88. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 89-94. In some embodiments, VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the amino acid sequences of SEQ ID NO: 95, 96 (or any one of 101-104), 97, 98, 99, and 100. In some embodiments, VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 105-110. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 111-116.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 117-122. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 123, 124 or 129, 125, 126, 127, and 128. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 130-135. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 136-141. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 142-147.

In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 148-153. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 154-159. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each contain the amino acid sequences of SEQ ID NO: 160, 161 or 166, 162, 163, 164, and 165. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 167, 168, or 173, 169, 170, 171, and 172, respectively. In some embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 174, 175, or 180, 176, 177, 178, and 179, respectively.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:5, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:6, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:6, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:11, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:11, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:12, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:13, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:14, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:17, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:18, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:19, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:20, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:20, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:25, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:25, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:26, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:26, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:27, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:27, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:28, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:28, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:29, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:29, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:30, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:30, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:31, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:31, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:32, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:32, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:35, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:35, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:36, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:36, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:37, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:37, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:38, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:38, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, VH comprises the amino acid sequence of SEQ ID NO:39, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:39, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO:40, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:40, while retaining the corresponding VL CDR or its PTM de-risked form.

In some embodiments, antibody and antigen-binding fragments comprising CDR sequences derived from the present disclosure are also provided, wherein the CDR sequences have one, two, or three amino acid substitutions, deletions, and/or additions.

Antibody-drug conjugates

In some implementations, the antibody or fragment may be conjugated with a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

In one embodiment, the antibody or fragment of this disclosure is covalently linked to a pharmaceutical moiety. The pharmaceutical moiety may be, or may be modified to include groups that react with coupling sites on the antibody. For example, the pharmaceutical moiety may be linked by alkylation (e.g., at the N-terminus of an ε-aminolysine residue or an antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation of an amino or carboxyl group, and coupling with a thiol.

In some embodiments, the number p of pharmaceutical moieties conjugated to each antibody molecule ranges from an average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3.

For example, when the chemical activation of a protein leads to the formation of a free thiol group, the protein can conjugate with a thiol-based reactant. In one aspect, this reagent is essentially specific to the free thiol group. Such reagents include, for example, maleimides, haloacetamides (e.g., iodine, bromine, or chlorine), haloesters (e.g., iodine, bromine, or chlorine), halomethyl ketones (e.g., iodine, bromine, or chlorine), benzyl halides (e.g., iodides, bromides, or chlorides), vinyl sulfones, and pyridine thiols.

Drugs can be linked to antibodies or fragments via linkers. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are generally readily cleavable under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline linker, a phenylalanine-lysine linker, or a maleimide hexanoate-valine-citrulline-p-aminobenzyloxycarbonyl linker (mc-Val-Cit-PABA). Another linker is sulfosuccinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid ester (smcc). Sulfon-smcc coupling occurs via a maleimide group that reacts with a thiol (thiol, -SH), while its sulfon-NHS ester is reactive to primary amines (such as those found in lysine and the N-terminus of proteins or peptides). Another type of linker is maleimide hexanoyl (MC). Other suitable linkers include those that are hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Other suitable cleavable linkers include disulfide linkers. Linkers can be covalently bound to antibodies to the extent that the antibody must degrade within the cell to release the drug, such as MC linkers.

The linker may include a group for binding to the antibody. For example, the linker may include an amino, hydroxyl, carboxyl, or thiol reactive group (e.g., maleimide, haloacetamide (e.g., iodine, bromine, or chlorine), haloester (e.g., iodine, bromine, or chlorine), halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodide, bromide, or chloride), vinyl sulfone, and pyridine thio.

In some embodiments, the drug component is a cytotoxic agent or cell inhibitor, immunosuppressant, radioactive isotope, toxin, etc. The conjugate can be used to inhibit the proliferation of tumor cells or cancer cells, induce apoptosis in tumor or cancer cells, or treat cancer in patients. Therefore, the conjugate can be used in various settings for treating cancer in animals. The conjugate can be used to deliver drugs to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds to or associates with cancer cells expressing CLDN6, and the conjugate and/or drug can be taken up within tumor cells or cancer cells via receptor-mediated endocytosis.

In some embodiments, the pharmaceutical part is maytansine or auristatin. In some embodiments, the pharmaceutical part is a macrocyclic ketone analog, such as eribulin. In some embodiments, the pharmaceutical part is a topoisomerase inhibitor, such as eczema and eczema derivatives.

Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in the connector) are cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in the release of the drug. The released drug then migrates freely within the cell and induces cytotoxicity, cell inhibition, or other activities. In some embodiments, the drug cleaves from antibodies outside the tumor cells or cancer cells, and the drug subsequently penetrates the cell or acts on the cell surface.

Examples of drug portions or payloads are selected from: eribulin (2-(3-amino-2-hydroxypropyl)hexacosahedrohydro-3-methoxy-26-methyl-20,27-bis(methylene)11,15-18,21-24,28-tricyclooxy-7,9-ethylene-12,15-methylene-9H,15H-furano(3,2-i)furano(2',3'-5,6)pyrano(4,3-b)(1,4)dioxane-5-(4H)-one), DM1 (maytansin, N2'-deacetylated-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetylated-N 2'-(3-mercapto-1-oxopropyl)-Maytansine), mc-MMAD (6-maleimide hexanoyl-monomethylaurestatin-D or N-methyl-L-valine-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valine), mc-MMAF (maleimide hexanoyl-monomethyl-4-methyl-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valine), mc-MMAF (maleimide hexanoyl-2-methyl ... Acyl-monomethylaurestatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-1-oxohexyl]-N-methyl-L-valine-L-valine-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropionyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimide hexanoyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylaurestatin E or N-[[[4-[[N-[6- (2,5-Dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-1-oxohexyl]-L-valine-N5-(aminocarbonyl)-L-guanyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valine-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrroloalkyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valineamide). DM1 is a derivative of the microtubule inhibitor maytansine, while MMAD, MMAE, and MMAF are derivatives of auristatin. In some implementations, the drug component is selected from mc-MMAF and mc-Val-Cit-PABA-MMAE.

Antibodies or fragments may be conjugated or fused with therapeutic agents, which may include detectable markers such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutics or diagnostics, cytotoxic agents (which may be drugs or toxins), ultrasound enhancers, non-radiolabels, combinations thereof, and other such agents known in the art.

Antibodies can be detectably labeled by conjugation to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding peptide is then determined by detecting the presence of light emitted during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalates.

Antibodies can also be detectably labeled using fluorescently emitting metals such as ^Eu152 or other lanthanides. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The technique of conjugating various components to antibodies is well known; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-256 (Alan R. Liss, Inc. (1985)); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd edition), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-653 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, and Future Prospective Of "The Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-316 (1985); and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Immunol Rev. (Vol. 52: pp. 119-158 (1982)).

It should be understood that any antibody or antigen-binding fragment of this disclosure is suitable for inclusion in the currently disclosed antibody-drug conjugates (ADCs). In one embodiment, the antibody or fragment comprises any one of antibodies 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, or 95F10, or their PTM derisked or affinity-matured forms of VH CDR and VLCDR.

In some embodiments, the antibody or fragment includes the VH CDR and VL CDR of any antibody in segment A (14G12, 393E9, and 113H5). In some embodiments, the antibody or fragment includes the VH CDR and VL CDR of any antibody in segment B (159D5, 24F10, and 493E10). In some embodiments, the antibody or fragment includes the VH CDR and VL CDR of any antibody in segment C (257F1, 353H11, 367B8, 389G2, and 109H7). In some embodiments, the antibody or fragment includes the VH CDR and VL CDR of any antibody in segment D (286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10).

In some embodiments, the antibody or antigen-binding fragment of the ADC includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:41; VH CDR2 contains the amino acid sequence of SEQ ID NO:42; VH CDR3 contains the amino acid sequence of SEQ ID NO:43; VL CDR1 contains the amino acid sequence of SEQ ID NO:44; VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and VL CDR3 contains an amino acid sequence selected from SEQ ID NO:46.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of SEQ ID NO:42; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:264. In some embodiments, VH CDR2 is de-risked by PTM. In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO:41; VH CDR2 contains the amino acid sequence of SEQ ID NO:42; VHCDR3 contains the amino acid sequence of SEQ ID NO:43; VL CDR1 contains the amino acid sequence of SEQ ID NO:44; VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and VL CDR3 contains an amino acid sequence selected from SEQ ID NO:265.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46, 264, and 265.

Exemplary VH sequences include amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. Exemplary VL sequences include amino acid sequences selected from SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:263. Yet another exemplary VH sequence includes an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:263.

In some embodiments, VH includes the amino acid sequence of any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, while retaining the corresponding VL CDR or its PTM de-risked form or affinity-matured form. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:197, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:262, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:262, while retaining the corresponding VL CDR or its affinity mature form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:263, while retaining the corresponding VL CDR or its affinity mature form.

Multifunctional molecules

Multifunctional molecules include antibodies or antigen-binding fragments specific to 5T4, such as those disclosed herein, and one or more antibodies or antigen-binding fragments specific to a second antigen.

In some implementations, the second antigen is a protein expressed on immune cells such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

In some implementations, the second antigen is CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

Different forms of bispecific antibodies are also provided. In some embodiments, each anti-5T4 fragment and the second fragment are independently selected from Fab fragments, single-chain variable fragments (scFv), or single-domain antibodies. In some embodiments, the bispecific antibody also includes an Fc fragment.

Bifunctional molecules that include not only antibodies but also antigen-binding fragments are also provided. As tumor antigen-targeting molecules, antibodies or antigen-binding fragments specific to 5T4, such as those described herein, can optionally be combined with immune cytokines or ligands via peptide linkers. The linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. These bifunctional molecules can combine immune checkpoint blockade effects with local immunomodulation at the tumor site.

In some embodiments, the anti-5T4 fragment includes the VHCDR and VL CDR of any antibody in segment A (14G12, 393E9, and 113H5). In some embodiments, the anti-5T4 fragment includes the VH CDR and VL CDR of any antibody in segment B (159D5, 24F10, and 493E10). In some embodiments, the anti-5T4 fragment includes the VH CDR and VL CDR of any antibody in segment C (257F1, 353H11, 367B8, 389G2, and 109H7). In some embodiments, the anti-5T4 fragment includes the VH CDR and VLCDR of any antibody in segment D (286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10).

In some embodiments, the anti-5T4 fragment includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:41; VH CDR2 contains the amino acid sequence of SEQ ID NO:42; VH CDR3 contains the amino acid sequence of SEQ ID NO:43; VL CDR1 contains the amino acid sequence of SEQ ID NO:44; VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and VL CDR3 contains an amino acid sequence selected from SEQ ID NO:46.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of SEQ ID NO:42; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:264. In some embodiments, VH CDR2 is de-risked by PTM. In some embodiments, VH CDR1 contains the amino acid sequence of SEQ ID NO:41; VH CDR2 contains the amino acid sequence of SEQ ID NO:42; VHCDR3 contains the amino acid sequence of SEQ ID NO:43; VL CDR1 contains the amino acid sequence of SEQ ID NO:44; VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and VL CDR3 contains an amino acid sequence selected from SEQ ID NO:265.

In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:41; VH CDR2 comprises the amino acid sequence of any one of SEQ ID NO:42 or SEQ ID NO:47-53; VH CDR3 comprises the amino acid sequence of SEQ ID NO:43; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:45; and VL CDR3 comprises an amino acid sequence selected from SEQ ID NO:46, 264, and 265.

Exemplary VH sequences include amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. Exemplary VL sequences include amino acid sequences selected from SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence includes amino acid sequences selected from SEQ ID NO:1, 181-184, 189-192, and 195-204. An exemplary VL sequence includes an amino acid sequence selected from SEQ ID NO:263. Yet another exemplary VH sequence includes an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:262. Another exemplary VH sequence comprises an amino acid sequence selected from SEQ ID NO:197. An exemplary VL sequence comprises an amino acid sequence selected from SEQ ID NO:263.

In some embodiments, VH includes the amino acid sequence of any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:1, 181-184, 189-192 and 195-204, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:2, 185-188, 193-194, 205-214, and 262-263, while retaining the corresponding VL CDR or its PTM de-risked form or affinity-matured form. In some embodiments, VH comprises the amino acid sequence of any one of SEQ ID NO:197, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:197, while retaining the corresponding VH CDR or its PTM de-risked form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:262, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:262, while retaining the corresponding VL CDR or its affinity mature form. In some embodiments, VL comprises the amino acid sequence of any of SEQ ID NO:263, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:263, while retaining the corresponding VL CDR or its affinity mature form.

Chimeric antigen receptor

In one embodiment, a chimeric antigen receptor (CAR) comprising an antibody or a fragment thereof of the present disclosure as a targeting unit is also provided. In some embodiments, the CAR comprises an antibody or a fragment thereof of the present disclosure, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain.

The transmembrane domain can be designed to fuse to an extracellular domain containing an antibody or fragment, optionally via a hinge domain. It can similarly fuse to an intracellular domain, such as a co-stimulatory domain. In some embodiments, the transmembrane domain may comprise a native transmembrane region of a co-stimulatory domain (e.g., the TM region of CD28T or 4-1BB used as a co-stimulatory domain) or a native transmembrane domain of a hinge domain (e.g., the TM region of CD8α or CD28T used as a hinge domain).

In some embodiments, a transmembrane domain may include a sequence that crosses the cell membrane but extends into the cytoplasm and/or into the extracellular space. For example, a transmembrane domain may include a transmembrane sequence that may further contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and/or extracellular space. Thus, a transmembrane domain includes a transmembrane region and may further include amino acids that extend beyond the inner or outer surface of the membrane itself; such sequences may still be considered "transmembrane domains".

In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably 2 to 10 amino acids in length, can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine duplexes (GS), glycine-serine-glycine triplets (GSG), or alanine-alanine-alanine triplets (AAA) provide suitable linkers.

In some embodiments, the CAR also includes a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Exemplary co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (... CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (α chain associated with B-cell antigen receptor complex), CD79B (β chain associated with B-cell antigen receptor complex), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD15 8F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(L IGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1 ), CD314(KG2D), CD319(SLAMF7), CD335(K-p46), CD336(K-p44), CD337(K-p30), CD3 52 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF 18), inducible T cell costimulators (ICOS), LFA-1 (CD 1la/CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1/CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments thereof, or combinations thereof.

In some embodiments, the cytoplasmic portion of the CAR also includes a signal transduction/activation domain. In one embodiment, the signal transduction/activation domain is a CD3ξ domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the CD3ξ domain.

Polynucleotides, mRNA, and methods for expressing or preparing antibodies

This disclosure also provides polynucleotide or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs of this disclosure. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, its variants or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of this disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, its variants or derivatives, either on the same polynucleotide molecule or on separate polynucleotide molecules.

In some implementations, the polynucleotide is an mRNA molecule. In some implementations, the mRNA can be introduced into target cells to express an antibody or a fragment thereof.

mRNA can be synthesized using any of the various known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). In short, IVT typically involves a linear or circular DNA template containing a promoter, a library of ribonucleoside triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitors. The exact conditions will vary depending on the specific application.

In some implementations, a DNA template is transcribed in vitro to prepare mRNA encoding an antibody. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence for encoding the desired antibody (e.g., encoding a heavy or light chain) and a termination signal.

The mRNA sequence encoding a desired antibody (e.g., encoding a heavy or light chain) can be determined and incorporated into a DNA template using standard methods. For example, virtual reverse translation is performed based on a degenerate genetic code, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence). Optimization algorithms can then be used to select appropriate codons. Typically, the G/C ratio is optimized to achieve the highest possible G/C ratio, while the frequency of tRNA is considered as much as possible based on codon usage. The optimized RNA sequence can be constructed and displayed, for example, using appropriate display equipment, and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate the stable and destabilizing properties or regions of the RNA separately.

mRNA can be synthesized as unmodified or modified mRNA. Typically, mRNA is modified to enhance stability. Modifications to mRNA may include, for example, modifications to RNA nucleotides. Modified mRNA may therefore include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding an antibody (e.g., mRNA encoding both heavy and light chains) may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives as purines and pyrimidines, such as, for example, 1-methyl-adenine. 2-Methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5- Uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2 -Thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine and aminophosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-dezoguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to those skilled in the art, for example from U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

In some implementations, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are chemical modifications to the phosphate groups of the nucleotide backbone contained in the RNA. Exemplary backbone modifications typically include, but are not limited to, modifications from methylphosphonates, methylaminophosphates, aminophosphates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), borophosphates, positively charged guanidinyl groups, etc., meaning that the phosphodiester bonds are replaced with other anionic, cationic, or neutral groups.

In some implementations, the mRNA (e.g., mRNA encoding both the heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars in nucleotides, including but not limited to those selected from 2'-deoxy-2'-fluoro-oligonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyl oligonucleotides... Sugar modification of ribonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligonucleotides and their isomers (2'-arylcytidine 5'-triphosphate, 2'-aryluridine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

In some implementations, mRNA (e.g., mRNA encoding the heavy and light chains) may contain base modifications of nucleotides (base modifications). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine nucleotide 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, and 5-methyluridine 5'-triphosphate. 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deadenosine 5'-triphosphate, 7-deadenosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate, or flavin 5'-triphosphate.

Typically, mRNA synthesis involves adding a "cap" at the N-terminus (5') and a "tail" at the C-terminus (3'). The presence of the cap is crucial for providing resistance to nucleases found in most eukaryotic cells. The presence of the tail protects the mRNA from degradation by exonucleases.

Therefore, in some implementations, the mRNA (e.g., mRNA encoding both the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate ester via guanylate transferase, creating a 5'5'5 triphosphate bond; then, 7-nitromethylation of guanine is performed using a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(A) tail. The poly(A) tail at the 3' end of the mRNA typically comprises about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding an antibody (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(C) tail. A suitable poly-C tail at the 3' end of mRNA typically contains about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to or replace the poly-A tail.

In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' and/or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region may be about 50 to 500 nucleotides in length (e.g., about 50 to 400 nucleotides, about 50 to 300 nucleotides, about 50 to 200 nucleotides, or about 50 to 100 nucleotides).

In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) contains a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the signal peptide coding sequence is directly or indirectly linked to the heavy or light chain coding sequence at the N-terminus.

This technology can be used to deliver any antibody known in the art and antibodies against a desired antigen that can be generated using standard methods. This invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human antibodies or humanized antibodies, chimeric antibodies, or bispecific antibodies.

Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide of this disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, the entire contents of which are incorporated herein by reference.

Treatment and Uses

As described herein, the antibodies, variants, or derivatives disclosed herein may be used in certain therapeutic and diagnostic approaches.

This disclosure also relates to antibody-based therapies involving the administration of antibodies or fragments of this disclosure to patients, such as animals, mammals, and humans, for the treatment of one or more of the conditions or illnesses described herein. Therapeutic compounds of this disclosure include, but are not limited to, antibodies of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of this disclosure (including variants and derivatives thereof as described herein).

The antibodies disclosed herein can also be used to treat or inhibit cancer. As mentioned above, 5T4 is rarely expressed in normal adult tissues, but is present at high levels in the placenta and most common tumors, typically accounting for more than 80% in kidney cancer, breast cancer, colon cancer, prostate cancer, and ovarian cancer.

Therefore, in some embodiments, a method for treating cancer in patients in need is provided. In one embodiment, this method involves administering an effective amount of the disclosed antibody or fragment or antibody-drug conjugate to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses 5T4.

This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. Suitable cells transduced with a vector encoding or in contact with a CAR, the CAR comprising the anti-5T4 antibody of this disclosure (or alternatively, engineered to express the anti-5T4 antibody of this disclosure), can be used. After such contact or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. The cells (e.g., T cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

In some implementations, the cells are isolated from the cancer patient themselves. In other implementations, the cells are provided by a donor or cell bank. When cells are isolated from a cancer patient, unwanted immune responses can be minimized.

Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some implementations, the cancer is one or more of stomach cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

The antibodies or variants or derivatives thereof disclosed herein may be used to treat, prevent, diagnose, and/or prognose other diseases or conditions associated with increased cell survival, including but not limited to the progression and/or metastasis of malignancies and related conditions such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, and liposarcoma. Tumors, chondrosarcomas, osteosarcomas, chordomas, angiosarcomas, endothelial sarcomas, lymphangiomas, lymphangioendothelial sarcomas, synovial tumors, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

The specific dosage and treatment regimen for any particular patient will depend on a number of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex and diet, timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The healthcare professional's judgment of these factors is within the realm of general technical skill in this field. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art.

Methods of administration for antibodies or fragments include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding peptides or compositions can be administered via any convenient route, such as by infusion or bolus, absorption through the epithelial or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, or transdermal patch), sublingually, or as oral or nasal sprays.

As used in this article, the term "parenteral" refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

Administration can be systemic or local. Furthermore, it may be desirable to introduce the antibodies of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be facilitated, for example, by an intraventricular catheter connected to a reservoir such as the Ommaya reservoir. Pulmonary administration may also be used, for example, by using an inhaler or nebulizer, and in combination with a nebulizer formulation.

It may be necessary to apply the antigen-binding peptides or compositions of this disclosure topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion during surgery, local application (e.g., in conjunction with postoperative wound dressings), by injection, via catheter, via suppository, or via implants that are porous, non-porous, or gel-like materials, including membranes such as sialic acid membranes or fibers. Preferably, when applying the proteins (including antibodies) of this disclosure, care must be taken to use materials that are not absorbed by the protein.

The amount of antibody or fragment or antibody-drug conjugate disclosed herein that will be effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, symptoms, or conditions can be determined using standard clinical techniques. Additionally, in vitro assays may be optionally employed to help identify the optimal dose range. The precise dose used in the formulation will also depend on the route of administration and the severity of the disease, symptom, or condition, and should be determined based on the physician's judgment and the individual patient's situation. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

As a general recommendation, the dosage of the antibodies or fragments disclosed herein administered to patients is typically from 0.001 mg/kg to 100 mg/kg of patient body weight, 0.01 mg/kg to 20 mg/kg of patient body weight, or 0.5 mg/kg to 10 mg/kg of patient body weight. Generally, due to the immune response to foreign peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses and less frequent administration of human antibodies are generally possible. Furthermore, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by modifying (e.g., lipidation) to enhance antibody uptake and tissue penetration (e.g., into the brain).

In another embodiment, the compositions of this disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of this disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

In another embodiment, the compositions disclosed herein are administered in combination with other treatment or preventative measures (e.g., radiotherapy).

Composition

This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody or fragment or antibody-drug conjugate and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

In one specific implementation, the term "pharmaceutically acceptable" means a substance approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more specifically, in humans. Furthermore, "pharmaceuticalally acceptable carriers" generally refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

The term "carrier" refers to a diluent, adjuvant, excipient, or solvent that is administered with the therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and tonic modulators, such as sodium chloride or glucose, may also be considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories using conventional binders and carriers (such as triglycerides). Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in E.W. Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of carrier to provide a form suitable for appropriate administration to the patient. The formulation should be suitable for the mode of administration. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to conventional methods to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also contain a solubilizer and a local anesthetic (such as lidocaine) to relieve pain at the injection site. These components are typically provided individually or in combination in unit dosage forms, for example, as lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, a sterile ampoule of water for injection or saline can be provided to mix the components prior to administration.

The compounds disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts that form with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts that form with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Example

Example 1: Production of mouse monoclonal antibodies against human 5T4

This embodiment describes the use of hybridoma technology to generate anti-human 5T4 mouse monoclonal antibodies.

Antigens: Human 5T4-His protein and CHO-K1 expressing human 5T4.

Immunization: To generate mouse monoclonal antibodies targeting human 5T4, SJL, Balb/C, and C57BL/6 mice were first immunized with 5T4-His protein. The immunized mice were then boosted with either 5T4-His protein or CHO-K1 expressing human 5T4. To select mice producing antibodies binding to 5T4 protein, antibody titers in the serum of immunized mice were assessed by ELISA and FACS. Briefly, microtiter plates were coated overnight at 4°C with 100 μL/well of 0.5 μg/mL or 1 μg/mL human 5T4 or cynomolgus monkey 5T4 protein in ELISA coating buffer, followed by blocking with 150 μL/well of 1% BSA. Serum dilution from immunized mice was added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed at OD 450 nm using a spectrophotometer. The immune response against the CHOK1-hu5T4 cell line was also tested using serum FACS, with the CHOK1 parental cell line used as a negative control. The resulting mice were used for fusion. The hybridoma supernatant was screened by ELISA.

Cell fusion: Fusion is performed via electrofusion. Fusted cells are seeded into 50 96-well plates for each fusion.

Screening: Supernatants targeting recombinant human (rh) 5T4-His protein and recombinant cynomolgus monkey 5T4-His protein were screened by ELISA. Then, the positive supernatants from the initial screening were confirmed by FACS combined with cell lines targeting CHOK1-hu5T4 and by ELISA-bound protein.

Subcloning and screening: Positive primary clones from each fusion were subcloned using a limiting dilution method to ensure that the subclones originated from a single parental cell. Subclones were screened using the same methods as primary clones, and the culture supernatant of positive clones underwent additional confirmatory screening by affinity sorting.

Hybridoma clones 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10, and 95F10 were selected for further analysis. The amino acid sequences of the variable regions of these clones are listed in Table 1 below. Tables 1-1 to 1-20 include, in addition to the original CDR sequences from these mouse antibodies, forms in which potential post-translational modifications (PTMs) have been removed or affinity-matured forms.

Table 1. Sequences of the variable regions of the selected clones

Table 1-1.14 CDR Sequences of G12

Table 1-2.393E9 CDR Sequence

Table 1-3.113H5 CDR sequence

113H5 sequence SEQ ID NO: CDRH1 SGYYWN 64 CDRH2 YINSDGSNNYNPSLKN 65 CDRH3 EEYDYWFAY 66 CDRL1 KASQNVGTNVA 67 CDRL2 SASNRYS 68 CDRL3 QQYNSYPYT 69

Table 1-4.159D5 CDR sequence

Table 1-5.24 CDR Sequences of F10

24F10 sequence SEQ ID NO: CDRH1 DYGMH 77 CDRH2 YISSGTSTIYYTDTVKG 78 CDRH3 GGDGYYRSTMDY 79 CDRL1 RASQSVSSSSYSYMH 80 CDRL2 SASNLAS 81 CDRL3 QHSWEVPRT 82

Table 1-6.493E10 CDR Sequence

Table 1-7.257F1 CDR Sequences

257F1 sequence SEQ ID NO: CDRH1 SYGLN 89 CDRH2 EIYPRSENTHYNEKFKG 90 CDRH3 GDWDFDH 91 CDRL1 SASSSVSYMN 92 CDRL2 GISNLAS 93 CDRL3 QQRSSYPRT 94

Table 1-8.353H11 CDR sequence

Table 1-9.367B8 CDR Sequence

367B8 sequence SEQ ID NO: CDRH1 SYWMH 105 CDRH2 NINPSNGNTNYNENFKS 106 CDRH3 GGWDFDY 107 CDRL1 SASSSVRYIH 108 CDRL2 DTSKLTS 109 CDRL3 QQWTSKPPWT 110

Table 1-10.389G2 CDR sequence

389G2 sequence SEQ ID NO: CDRH1 SYWMH 111 CDRH2 NINPSNGNTNYNERFKS 112 CDRH3 GGWDFDY 113 CDRL1 SASSSVRYIH 114 CDRL2 DTSKLTS 115 CDRL3 QQWTSKPPWT 116

Table 1-11.109H7 CDR sequence

CDR sequences of Table 1-12.286B4

Table 1-13.37G6 CDR sequence

37G6 sequence SEQ ID NO: CDRH1 SYWIT 130 CDRH2 DIYPGSGTINYNEKFKS 131 CDRH3 SDGNYYFDY 132 CDRL1 RASSSVNYMH 133 CDRL2 ATSNLAS 134 CDRL3 QQWSSNPPT 135

CDR sequences of Table 1-14.267B5

267B5 sequence SEQ ID NO: CDRH1 SSYYWN 136 CDRH2 YISYDGSNNYNPSLKN 137 CDRH3 SWDGGYAMDN 138 CDRL1 KASQSVSNDVA 139 CDRL2 FASNRYT 140 CDRL3 QQDYTSPWT 141

Table 1-15.425G1 CDR Sequence

425G1 sequence SEQ ID NO: CDRH1 DTYMH 142 CDRH2 RIDPANGHTIFASKFQG 143 CDRH3 FTMVVVPWYFDV 144 CDRL1 KASQDVSTAVA 145 CDRL2 WASTRHT 146 CDRL3 QQHYSSPLT 147

Table 1-16.449H9 CDR sequence

449H9 sequence SEQ ID NO: CDRH1 NYWMN 148 CDRH2 QIYPGDGDTNYNGKFKN 149 CDRH3 HYDYPYYYAMDY 150 CDRL1 RASQDIGIALT 151 CDRL2 ATSSLDS 152 CDRL3 LQYIISPYT 153

Table 1-17.49C5 CDR sequence

Table 1-18.119G5 CDR sequence

Table 1-19.85B10 CDR Sequence

Table 1-20.95F10 CDR Sequence

Example 2: Binding affinity of mAbs

The binding of chimeric mAbs from these clones to the recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method via Biacore. The mAbs were captured using a protein A microarray. Serial dilutions of the human 5T4-his-tagged protein were injected onto the captured antibody at a flow rate of 30 μl/min for 2–3 minutes. Antigen dissociation was allowed for 360–1000 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software.

As shown in Table 2 below, most of the tested antibodies exhibited nanomolar or sub-nanomolar binding affinity to recombinant human 5T4 protein.

Table 2. Affinity measured by Biacore

ELISA assays were performed to evaluate the binding of the chimeric antibody to humans and cynomolgus monkeys, respectively. An antibody moiety targeting 5T4, natropumab (NeoTX) (abbreviated as natropumab in this disclosure), was generated and used as a baseline.

In short, microtiter plates were coated with 100 μl/well of PBS solution containing 1 μg/ml human and cynomolgus 5T4 protein, incubated overnight at 4°C, and then blocked with 150 μl/well of 1% BSA. Serial dilutions of the chimeric antibody were added to each well and incubated at RT for 1 h. The plates were washed with PBS/Tween and then incubated at RT for 30 min with a mouse anti-human IgGFc antibody conjugated with horseradish peroxidase (HRP). After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. All 5T4 chimeric mAbs bound to human and cynomolgus 5T4 (Figures 1-2 and Table 3). Naptomomumab showed very weak cross-activity against cynomolgus 5T4.

Example 3: Binding activity against 5T4 antigen

3.1 Interspecies activity

Table 3. Interspecies activity of clones as assessed by ELISA

3.2 Epitope partitioning via competitive ELSA

Competitive ELISA was performed to classify the tested 5T4 mAbs based on their binding epitopes to human 5T4.

In short, 100 μl/well of a PBS solution containing 0.5 μg/ml human 5T4 protein was used to coat microtiter plates, which were incubated overnight at 4°C, and then blocked with 150 μl/well of 1% BSA. Serial dilutions of the chimeric antibody and 0.2 μg/ml biotin-conjugated reference mAb were added to each well, and the plates were incubated at RT for 1 hour. The plates were washed with PBS/Tween and then incubated with streptavidin-HRP at RT for 15 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. Based on their competition performance with the reference mAb, the 5T4 mAb was divided into four segments (segments A-D), as shown in Figures 3-8 and Table 4.

Table 4. Tablet Categories

category Exemplary clone Section A Napotumomab, 14G12, 393E9, 113H5 Section B 159D5,24F10,493E10 Section C 257F1,353H11,367B8,389G2,109H7 Section D 286B4,37G6,267B5,425G1,449H9,49C5,119G5,85B10,95F10

3.3 FACS Test

Cell-based binding: FACS was used to evaluate the binding activity of all chimeric mAbs for CHO-K1 (CHOK1-hu5T4) expressing human 5T4.

In short, CHOK1-hu5T4 cells were washed with FACS buffer and aliquoted with serially diluted 5T4 chimeric mAbs at 4°C for 30 minutes in each well. After washing with FACS buffer, PE goat anti-human IgG Fc secondary antibody (eBioscience ^™ , Invitrogen) was added to each well and incubated at 4°C for 30 minutes. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was assessed using a MACSQuant analyzer 16. As shown in Figure 9, all tested 5T4 chimeric mAbs bound to CHOK1-hu5T4 cells, and antibodies in different segments showed different binding activities.

Example 4.14 Humanization of G12

Humanized mAbs were generated using the 14G12 variable region gene. In the first step of this process, the amino acid sequences of VH and VK of the 14G12 gene were compared with available human Ig gene sequence databases to find the best-matching human germline Ig gene sequence overall.

The germline sequences used for CDR transplantation and the resulting humanized sequences are listed in Table 5.

Table 5-1.14 Humanization of G12 - Round 1

Table 5-2. Humanized antibodies from 14G12—Round 1

Table 5-3.14 Humanization of G12—Round 2

Table 5-4. Humanized antibodies from 14G12—Round 2

Table 5-5.14 Humanization of G12—Round 3

Table 5-6. Humanized antibodies from 14G12—Round 3

Example 5: Binding activity of 14G12 humanized antibody against human 5T4 antigen

This embodiment tested the binding activity of the 14G12 humanized antibody to human 5T4 protein.

5.1 Binding of 14G12 humanized antibody to 5T4 ELISA

To evaluate the binding activity of the 14G12 humanized antibody to human 5T4, the 14G12 chimeric antibody, together with the 14G12 humanized mAb, was subjected to ELISA testing.

In short, microtiter plates were coated overnight at 4°C with 100 μl/well of PBS solution containing 1 μg/ml human 5T4-His protein, followed by blocking with 150 μl/well of 1% BSA. Serial dilutions of the antibody were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG-HRP at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. As shown in Figure 10 and Table 6, most of the humanized antibodies bound human 5T4 with high activity.

Table 6. Binding activity of humanized 14G12 antibody against human 5T4

5.2 Cell-based binding of 14G12 humanized antibody to 5T4

To evaluate the cell-based binding properties of the 14G12 humanized antibody to human 5T4 cells, the antibody was tested in CHOK1-hu5T4 cells by FACS analysis. CHOK1-hu5T4 cells, totaling 1 × ^10⁵ cells per well, were incubated with serially diluted antibody in FACS buffer at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibody conjugated to PE was added to each well and incubated at 4°C for 30 min. Following washing, the MFI of PE was evaluated using a MACSQuant 16 analyzer. As shown in Figure 11, some of the listed 14G12 humanized antibodies exhibited binding abilities comparable to the 14G12 chimeric antibody.

5.3 Ranking of protein affinity of 14G12 humanized antibody for 5T4

The binding of 14G12 humanized antibody to recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method on Biacore. The mAb was captured using a protein A microarray. Serial dilutions of the human 5T4-his tag protein were injected onto the captured antibody at a flow rate of 30 μl/min for 120–180 seconds. Antigen dissociation was allowed for 360–1000 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Table 7 below.

Table 7. Affinity ranking as measured by Biacore

Example 6. Humanization of the 393E9 antibody

The 393E9 variable region gene was used to generate humanized mAbs. In the first step of this process, the amino acid sequences of VH and VK of 393E9 were compared with available human Ig gene sequence databases to find the best-matching human germline Ig gene sequence overall.

The germline sequences used for CDR transplantation and the resulting humanized sequences are listed in Table 8.

Humanization of Table 8.393E9

Table 8-2. Humanized antibodies from 393E9

Example 7: Binding activity of 393E9 humanized antibody against human 5T4 antigen

This embodiment tested the binding activity of the 393E9 humanized antibody to human 5T4 protein.

7.1 393E9 humanized antibody binding to 5T4 ELISA

To evaluate the binding activity of the humanized 393E9 antibody to human 5T4, the 393E9 chimeric antibody (393E9-C) together with the humanized 393E9 mAb was tested by ELISA.

In short, microtiter plates were coated overnight at 4°C with 100 μl/well of PBS solution containing 1 μg/ml human 5T4-His protein, followed by blocking with 150 μl/well of 1% BSA. A four-fold dilution of antibody starting at 20 nM was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG-HRP at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. As shown in Figure 12 and Table 9, most of the humanized antibodies bound human 5T4 with high activity.

Table 9. Binding activity of humanized 393E9 antibody against human 5T4

7.2 Cell-based binding of 393E9 humanized antibody to 5T4

To evaluate the cell-based binding properties of the 393E9 humanized antibody to human 5T4 cells, the antibody was tested in CHOK1-hu5T4 cells by FACS analysis. In FACS buffer, 1 × ^10⁵ CHOK1-hu5T4 cells per well were incubated with serially diluted antibody at 3-fold concentrations starting from 50 nM at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibody conjugated to PE was added to each well and incubated at 4°C for 30 min. After washing, the MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figure 13, some of the listed 393E9 humanized antibodies exhibited binding abilities comparable to the 393E9 chimeric antibody.

7.3 Ranking of protein affinity of 393E9 humanized antibody for 5T4

The binding of the 393E9 humanized antibody to the recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method via Biacore. The mAb was captured using a protein A microarray. Serial dilutions of the human 5T4-his tag protein were injected onto the captured antibody at a flow rate of 30 μl/min for 3 minutes. Antigen dissociation was allowed for 280–800 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Table 10 below.

Table 10. Affinity ranking as measured by Biacore

Example 8. Humanization of the 159D5 antibody

The 159D5 variable region gene was used to generate humanized mAbs. In the first step of this process, the amino acid sequences of VH and VK of 159D5 were compared with available human Ig gene sequence databases to find the best-matching human germline Ig gene sequence overall.

The germline sequences used for CDR transplantation and the resulting humanized sequences are listed in Table 11.

Humanization of Table 11-1.159D5

Table 11-2. Humanized antibodies from 159D5

Example 9: Binding activity of 159D5 humanized antibody against human 5T4 antigen

This embodiment tested the binding activity of the 159D5 humanized antibody to human 5T4 protein.

9.1 159D5 humanized antibody binding to 5T4 ELISA

To evaluate the binding activity of the 159D5 humanized antibody to human 5T4, the 159D5 chimeric antibody, together with the 159D5 humanized mAb, was tested by ELISA.

In short, microtiter plates were coated overnight at 4°C with 100 μl/well of PBS solution containing 1 μg/ml human 5T4-His protein, followed by blocking with 150 μl/well of 1% BSA. Serial dilutions of the antibody were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG-HRP at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. As shown in Figure 14 and Table 12, most of the humanized antibodies bound human 5T4 with high activity.

Table 12. Binding activity of humanized 159D5 antibody against human 5T4

9.2 Cell-based binding of 159D5 humanized antibody to 5T4

To evaluate the cell-based binding properties of the 159D5 humanized antibody to human 5T4 cells, the antibody was tested in CHOK1-hu5T4 cells by FACS analysis. CHOK1-hu5T4 cells, totaling 1 × ^10⁵ cells per well, were incubated with serially diluted antibody in FACS buffer at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibody conjugated to PE was added to each well and incubated at 4°C for 30 min. Following washing, the MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figure 15, most of the listed 159D5 humanized antibodies exhibited binding capacity comparable to the 159D5 chimeric antibody.

9.3 Ranking of protein affinity of 159D5 humanized antibody for 5T4

The binding of the 159D5 humanized antibody to the recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method on the Biacore platform. The mAb was captured using a protein A microarray. 50 nM human 5T4-his tag protein was injected onto the captured antibody at a flow rate of 30 μl/min for 3 minutes. Antigen dissociation was allowed for 600 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Table 13 below.

Table 13. Affinity ranking as measured by Biacore

Example 10. Humanization of the 286B4 antibody

The 286B4 variable region gene was used to generate humanized mAbs. In the first step of this process, the amino acid sequences of VH and VK of 286B4 were compared with available human Ig gene sequence databases to find the best-matching human germline Ig gene sequence overall.

For the heavy chain of 286B4, VH4-28/JH6 was selected as the humanized backbone, while VL-O18/JK4 was the most suitable germline for the light chain. Humanized 286B4 CDR graft antibodies were then designed, with CDRL1, L2, and L3 grafted onto the VL-O18-JK4 framework sequence and CDRH1, H2, and H3 grafted onto the VH4-28-JH6 framework sequence. A 3D model was then generated to determine the amino acids crucial for antibody binding and conformation in the original mouse FR region sequence. Based on the 286B4 CDR graft antibody sequence, four additional humanized heavy chains and five additional light chains were created.

The germline sequences used for CDR transplantation and the resulting humanized sequences are listed in Table 14.

Humanization of Table 14-1.286B4

Table 14-2. Humanized antibodies from 286B4

Example 11: Binding activity of 286B4 humanized antibody against human 5T4 antigen

This embodiment tested the binding activity of the 286B4 humanized antibody to human 5T4 protein.

11.1 ELISA binding to 5T4

To assess the binding activity of the clone, the 286B4 chimeric antibody was tested together with the 286B4 humanized mAb by ELISA.

In summary, microtiter plates were coated overnight at 4°C with 100 μl/well of PBS solution containing 1 μg/ml human 5T4-His protein, followed by blocking with 150 μl/well of 1% BSA. A four-fold dilution of the antibody to be tested, starting at 20 nM, was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG-HRP at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. As shown in Figure 16 and Table 15, most humanized clones bind to human 5T4 with high potency.

Table 15. Binding activity of humanized 286B4 antibody against human 5T4

11.2 Cell-based binding of 5T4

To evaluate the cell-based binding properties of the 286B4 humanized antibody to human 5T4, the binding of the tested antibody to CHOK1-hu5T4 (a human 5T4 high-expressing cell line) or MCF-7 cells (a human 5T4 low-expressing cell line) was analyzed by FACS. In FACS buffer, 1 × ^10⁵ CHOK1-hu5T4 or MCF7 cells per well were incubated with serially diluted 4- or 3-fold antibodies starting at 50 nM at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibody conjugated to PE was added to each well and incubated at 4°C for 30 min. After washing, the MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figures 17A (CHO-hu5T4 cells) and 17B (MCF-7 cells), the 286B4-3/8/13/18/5/10/15 humanized antibody showed comparable binding ability to the 286B4 chimeric antibody.

11.3 Protein affinity ranking for human 5T4

The binding of the 286B4 humanized antibody to the recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method on the Biacore assay. The mAb was captured using a protein A microarray. Serial dilutions of the human 5T4-his tag protein were injected onto the captured antibody at a flow rate of 30 μl/min for 3 minutes. Antigen dissociation was allowed for 280–800 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Table 16 below.

Table 16. Affinity ranking as measured by Biacore

PTM Removal Confirmation for Examples 12.393E9, 159D5, and 286B4

12.1 Confirmation of PTM removal for 393E9

393E9 VH CDR2 includes NG and NS residues (Kabat number), which are at risk of post-translational modification (PTM) and pose a challenge to future manufacturing. Therefore, in this embodiment, NG on VH is mutated to NA and NS is mutated to YS to prevent PTM. The sequences of potential PTM removal sites are listed in Table 17.

Table 17-1: Humanization of 393E9 with potential PTM sites removed

Table 17-2. Humanized antibodies against 393E9 with potential PTM sites removed

To evaluate the binding properties of PTM-removed antibodies to surface 5T4, the binding of trispecific antibodies with anti-5T4 moieties of Hu393E9-45-P2, Hu393E9-53-P2, Hu393E9-62-P2, or chimeric 393E9 to CHOK1-hu5T4 cells was analyzed by FACS. A total of 1 × ^10⁵ cells per well was incubated with 4-fold serial dilutions of antibody starting at 100 nM at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibodies conjugated to PE were added to each well and incubated at 4°C for 30 min. The MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figure 18A, trispecific antibodies with anti-5T4 moieties of Hu393E9-45-P2 or Hu393E9-62-P2 showed enhanced binding ability to 5T4-expressing cells compared to trispecific antibodies with chimeric 393E9. The only difference between the three specific antibodies tested was the anti-5T4 moiety.

12.2 Confirmation of PTM removal for 159D5

The 159D5 VH CDR2 contains DS residues (Kabat numbered), which are at risk of post-translational modification (PTM) and pose a challenge for future manufacturing. Therefore, in this embodiment, the DS on VH is mutated to DA to prevent PTM. The sequences of potential PTM removal sites are listed in Table 18. As shown in Figures 14B, 15, and Table 13, the PTM-removing antibody of 159D5-P1 exhibits binding ability comparable to that of the 159D5 chimeric antibody of 159D5-C.

Table 18-1.159D5 CDRH2 PTM Removal

Table 18-2. Humanization of 159D5 with potential PTM sites removed

Table 18-3. Humanized 159D5 antibodies with potential PTM sites removed

12.3 Confirmation of PTM removal for 286B4

286B4 VH CDR2 includes DG residues (Kabat number), which are at risk of post-translational modification (PTM) and pose a challenge to future manufacturing. Therefore, in this embodiment, the DG on VH is mutated to DA to prevent PTM. The sequences of potential PTM removal sites are listed in Table 19.

Table 19-1. Humanization of 286B4 with potential PTM sites removed

Table 19-2: Humanized antibodies against 286B4 with potential PTM sites removed

To evaluate the binding properties of PTM-removed antibodies to surface 5T4, the binding of trispecific antibodies with the anti-5T4 motif of Hu286B4-3-P1, Hu286B4-5-P1, Hu286B4-8-P1, Hu286B4-15-P1, or a chimeric 286B4 fragment to CHOK1-hu5T4 cells was analyzed by FACS. A total of 1 × ^10⁵ cells per well was incubated with 4-fold serial dilutions of antibody starting at 100 nM at 4°C for 30 min. After washing with FACS buffer, anti-human IgG antibodies conjugated to PE were added to each well and incubated at 4°C for 30 min. The MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figure 18B, trispecific antibodies with the PTM-removed 286B4 fragment showed enhanced binding ability to 5T4-expressing cells compared to trispecific antibodies with a chimeric 286B4 fragment. The only difference between the tested trispecific antibodies was the anti-5T4 motif.

Example 13. Affinity maturation of humanized antibody Hu14G12-28

To enhance the affinity of the humanized Hu14G12-28 antibody, affinity maturation was performed. In short, four phage libraries containing single- or two-point saturation mutations in the CDR region of Hu14G12-28 were constructed. Through one round of solid- or liquid panning, two candidates with unique mutations in the CDR were obtained. The CDRs of these candidates were then grafted into the Hu14G12-28 framework to generate antibodies for further binding and affinity validation.

The amino acid sequences of the variable regions of the framework transplanted into affinity maturation candidates are listed in Table 20 below.

Table 20-1. Sequences of the variable regions of antibodies that mature with affinity.

Table 20-2. Affinity-matured antibodies from Hu14G12-28

Table 20-3. CDR sequence of Hu14G12-28-88#

Table 20-4. CDR sequence of Hu14G12-28-108#

13.1 ELISA binding to 5T4

To assess the binding activity of the affinity-matured antibody to human 5T4, Hu14G12-28, along with Hu14G12-28-88# and Hu14G12-28-108#, was tested by ELISA.

In short, microtiter plates were coated overnight at 4°C with 30 μl/well of PBS solution containing 2 μg/ml human 5T4-His protein, followed by blocking with 5% PBS/emulsion. Serial dilutions of the antibody were added to each well and incubated at room temperature for 1 hour. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG-HRP at room temperature for 50 minutes. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. As shown in Figure 19, the affinity-matured antibody bound to human 5T4 with higher potency than the parental Hu14G12-28 antibody.

13.2 Cell-based binding to 5T4

To evaluate the binding properties of affinity-matured antibodies to surface 5T4, the binding of the tested antibodies to CHOK1-hu5T4, HEK293-hu5T4, or MCF-7 cells was analyzed by FACS. A total of 1 × ^10⁵ cells per well was incubated with serially diluted antibodies (4- or 5-fold dilutions starting at 133 nM or 100 nM) at 4°C for 60 min. After washing with FACS buffer, anti-human IgG antibodies conjugated to PE were added to each well and incubated at 4°C for 30 min. The MFI of PE was evaluated using a MACSQuant analyzer 16. As shown in Figures 20A-20B (CHOK1-hu5T4 cells), 20C-20D (HEK293-hu5T4 cells), and 20E (MCF-7 cells), affinity-matured antibodies showed enhanced binding to 5T4-expressing cells compared to the parental Hu14G12-28 antibody.

13.3 Binding affinity to 5T4

The binding affinity of the antibody to the recombinant 5T4 protein (human 5T4-his tag) was tested using a capture method via the Biacore assay. The antibody was captured using a protein A chip. Serial dilutions of the human 5T4-his tag protein were injected onto the captured antibody at a flow rate of 30 μl/min for 3 minutes. Antigen dissociation was allowed for 300 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software.

As shown in Table 21 below, compared with the parental Hu14G12-28 antibody, the affinity-matured antibodies Hu14G12-28-88# and Hu14G12-28-108# significantly increased their binding affinity to human 5T4 protein by 9.45 times and 7.41 times, respectively, making them ideal anti-5T4 antibodies for antibody-drug conjugates (ADCs) and bispecific antibodies.

Table 21. Affinity measured by Biacore

This disclosure is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover various modifications and variations of this disclosure, provided that they fall within the scope of the appended claims or their equivalents.

All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims (33)

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment being specific for human 5T4 carcinoembryonic trophoblast glycoprotein (5T4) protein, and comprising a heavy chain variable region (VH) and a light chain variable region (VL), said heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, said light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein said VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 each comprise the following amino acid sequences: SEQ ID NO: 41, 42 (or any one of 47-53), 43, 44, 45 and 46 (or 264 or 265); SEQ ID NO: 54, 55 (or any one of 60-63 or 266 or 267), 56, 57, 58 and 59; SEQ ID NO:64-69; SEQ ID NO: 70, 71 (or 76), 72, 73, 74 and 75; SEQ ID NO:77-82; SEQ ID NO:83-88; SEQ ID NO:89-94; SEQ ID NO: 95, 96 (or any one of 101-104), 97, 98, 99, and 100; SEQ ID NO:105-110; SEQ ID NO:111-116; SEQ ID NO:117-122; SEQ ID NO: 123, 124 or 129, 125, 126, 127 and 128; SEQ ID NO:130-135; SEQ ID NO:136-141; SEQ ID NO:142-147; SEQ ID NO:148-153; SEQ ID NO:154-159; SEQ ID NO: 160, 161 or 166, 162, 163, 164 and 165; SEQ ID NO: 167, 168 or 173, 169, 170, 171 and 172; or SEQ ID NO: 174, 175 or 180, 176, 177, 178 and 179. 2. The antibody or its antigen-binding fragment according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:54; The VH CDR2 comprises an amino acid sequence selected from SEQ ID NO: 55, 60-63, and 266-267; The VH CDR3 contains the amino acid sequence of SEQ ID NO:56; The VL CDR1 contains the amino acid sequence of SEQ ID NO:57; The VL CDR2 contains the amino acid sequence of SEQ ID NO:58; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:59. 3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH comprises an amino acid sequence selected from SEQ ID NO:3, 215-221 and 248-256, and the VL comprises an amino acid sequence selected from SEQ ID NO:4 and 222-226. 4. The antibody or its antigen-binding fragment according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:123; The VH CDR2 contains an amino acid sequence selected from SEQ ID NO: 124 and 129; The VH CDR3 contains the amino acid sequence of SEQ ID NO:125; The VL CDR1 contains the amino acid sequence of SEQ ID NO:126; The VL CDR2 contains the amino acid sequence of SEQ ID NO:127; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:128. 5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the VH comprises an amino acid sequence selected from SEQ ID NO:23, 236-241 and 259-261, and the VL comprises an amino acid sequence selected from SEQ ID NO:24 and 242-247. 6. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:41; The VH CDR2 contains an amino acid sequence selected from SEQ ID NO:42 and 47-53; The VH CDR3 contains the amino acid sequence of SEQ ID NO:43; The VL CDR1 contains the amino acid sequence of SEQ ID NO:44; The VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:46, 264, or 265. 7. The antibody or antigen-binding fragment thereof according to claim 6, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:41; The VH CDR2 contains the amino acid sequence of SEQ ID NO:42; The VH CDR3 contains the amino acid sequence of SEQ ID NO:43; The VL CDR1 contains the amino acid sequence of SEQ ID NO:44; The VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:264 or 265. 8. The antibody or antigen-binding fragment thereof according to claim 6, wherein the VH comprises an amino acid sequence selected from SEQ ID NO:1, 181-184, 189-192 and 195-204, and the VL comprises an amino acid sequence selected from SEQ ID NO:2, 185-188, 193-194, 205-214 and 262-263. 9. The antibody or antigen-binding fragment thereof according to claim 7, wherein the VH comprises the amino acid sequence of SEQ ID NO: 197, and the VL comprises the amino acid sequence of SEQ ID NO: 262 or 263. 10. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:70; The VH CDR2 contains an amino acid sequence selected from SEQ ID NO: 71 and 76; The VH CDR3 contains the amino acid sequence of SEQ ID NO:72; The VL CDR1 contains the amino acid sequence of SEQ ID NO:73; The VL CDR2 contains the amino acid sequence of SEQ ID NO:74; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:75. 11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VH comprises an amino acid sequence selected from SEQ ID NO:7, 227-229 and 257, and the VL comprises an amino acid sequence selected from SEQ ID NO:8, 230-235 and 258. 12. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:95; The VH CDR2 contains an amino acid sequence selected from SEQ ID NO: 96 and 101-104; The VH CDR3 contains the amino acid sequence of SEQ ID NO:97; The VL CDR1 contains the amino acid sequence of SEQ ID NO:98; The VL CDR2 contains the amino acid sequence of SEQ ID NO:99; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:100. 13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO:15, and the VL comprises the amino acid sequence of SEQ ID NO:16. 14. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:117; The VH CDR2 contains the amino acid sequence of SEQ ID NO:118; The VH CDR3 contains the amino acid sequence of SEQ ID NO:119; The VL CDR1 contains the amino acid sequence of SEQ ID NO:120; The VL CDR2 contains the amino acid sequence of SEQ ID NO:121; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:122. 15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the VH comprises the amino acid sequence of SEQ ID NO:21, and the VL comprises the amino acid sequence of SEQ ID NO:22. 16. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:154; The VH CDR2 contains the amino acid sequence of SEQ ID NO:155; The VH CDR3 contains the amino acid sequence of SEQ ID NO:156; The VL CDR1 contains the amino acid sequence of SEQ ID NO:157; The VL CDR2 contains the amino acid sequence of SEQ ID NO:158; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:159. 17. The antibody or antigen-binding fragment thereof according to claim 16, wherein the VH comprises the amino acid sequence of SEQ ID NO:33, and the VL comprises the amino acid sequence of SEQ ID NO:34. 18. The antibody or fragment thereof according to any one of claims 1-17, wherein the antibody or fragment thereof is a bivalent Fab antibody, or a fragment selected from F(ab’)2, F(ab)2, Fab’, Fab, Fv and scFv. 19. An antibody-drug conjugate comprising an antibody or fragment thereof conjugated to a drug portion according to any one of claims 1-18. 20. The antibody-drug conjugate of claim 19, wherein the drug portion is a cytotoxic agent or a cell inhibitor. 21. The antibody-drug conjugate of claim 20, wherein the drug portion is maytansine, aurestatin, or a macrolide analog. 22. The antibody-drug conjugate of claim 21, wherein the drug portion comprises monomethylaurestatin E (MMAE) or monomethylaurestatin F (MMAF). 23. The antibody-drug conjugate according to any one of claims 19-23, wherein the drug portion is linked to the antibody or a fragment thereof via a hydrolyzable linker under acidic conditions. 24. The antibody-drug conjugate according to any one of claims 19-23, wherein: The VH CDR1 contains the amino acid sequence of SEQ ID NO:41; The VH CDR2 contains the amino acid sequence of SEQ ID NO:42; The VH CDR3 contains the amino acid sequence of SEQ ID NO:43; The VL CDR1 contains the amino acid sequence of SEQ ID NO:44; The VL CDR2 contains the amino acid sequence of SEQ ID NO:45; and The VL CDR3 contains the amino acid sequence of SEQ ID NO:264 or 265. 25. A multispecific antibody comprising an antigen-binding fragment according to any one of claims 1-18 and one or more antibodies or antigen-binding fragments having binding specificity to non-5T4 target antigens. 26. A chimeric antigen receptor (CAR), said CAR comprising an antigen-binding fragment according to any one of claims 1-8, a transmembrane domain, a co-stimulatory domain, and a CD3ξ intracellular domain. 27. One or more polynucleotides, said polynucleotides encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-18 or a CAR according to claim 26. 28. The polynucleotide of claim 27, wherein the polynucleotide is one or more mRNAs. 29. The polynucleotide of claim 28, wherein the mRNA is chemically modified. 30. A cell comprising a polynucleotide according to any one of claims 27-29. 31. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-18, an antibody-drug conjugate according to any one of claims 19-24, a multispecific antibody according to claim 25, a CAR according to claim 26, a polynucleotide according to any one of claims 27-29, or a cell according to claim 30, and a pharmaceutically acceptable carrier. 32. A method of treating cancer in a patient in need, the method comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-18, an antibody-drug conjugate according to any one of claims 19-24, a multispecific antibody according to claim 25, a CAR according to claim 26, a polynucleotide according to any one of claims 27-29, or a cell according to claim 30. 33. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-18, the antibody-drug conjugate according to any one of claims 19-24, the multispecific antibody according to claim 25, the CAR according to claim 26, the polynucleotide according to any one of claims 27-29, or the cell according to claim 30 in the preparation of a medicament for treating cancer.