KR20250128351A - Treatment of cancer using a combination of antibodies that bind to EGFR and cytotoxic drugs - Google Patents

Abstract

The present disclosure relates to the field of therapeutic antibodies for the treatment of subjects with cancer. More particularly, the present disclosure relates to the treatment of cancer using an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and/or analog thereof, in combination therapy further comprising administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38. The cancer may be head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer. The present disclosure also relates to pharmaceutical formulations, kit components, and dosage regimens.

Description

Treatment of cancer using a combination of antibodies that bind to EGFR and cytotoxic drugs

The present disclosure relates to means and methods for treating cancer. Specifically, the present disclosure relates to a method for treating cancer in a subject using a combination of an antibody that binds EGFR and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38. The present disclosure further relates to combinations for use in such methods and in the manufacture of medicaments for treating cancer.

Colorectal cancer (CRC) is a leading cause of death worldwide, partly due to increasing resistance to current treatments. While some novel CRC therapies have been developed, many have failed in clinical trials, and metastatic CRC remains largely incurable. The current standard treatment for advanced CRC in clinical practice is chemotherapy, which blocks essential cancer cell functions and kills dividing cells.

Several anti-EGFR drugs have demonstrated some level of efficacy in targeted therapy for metastatic CRC (mCRC). However, resistance to anti-EGFR therapy occurs for various reasons, such as the emergence of mutations that cause resistance or the presence of innate resistance to anti-EGFR therapy. Most patients with cancers sensitive to anti-EGFR therapy later develop resistant cancers.

Current standard treatments for CRC include oxaliplatin, fluorouracil, and irinotecan. For liver metastases, standard treatment is combined with EGFR or VEGF-blocking monoclonal antibodies.

Due to increasing resistance to current treatments, there remains a need to develop additional therapies targeting cancers such as CRC to improve cancer treatment or expand treatment options.

The present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to an extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to an extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering fluorouracil, TAS-102, oxaliplatin, venetoclax or SN-38.

The present disclosure demonstrates that combination therapy comprising administering a multispecific antibody targeting EGFR and LGR5 in combination with a fluoropyrimidine, a platinum-based chemotherapy agent, a BCL-2 inhibitor, or SN-38 is more effective compared to the effect of the multispecific antibody or the fluoropyrimidine, the platinum-based chemotherapy agent, the BCL-2 inhibitor, or SN-38 administered individually.

In certain aspects, the present disclosure further provides a combination of an antibody or a functional portion, derivative and/or analog thereof with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for use in the treatment of cancer.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering fluorouracil, oxaliplatin and folinic acid.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering fluorouracil, irinotecan and folinic acid.

Additionally provided is a method of treating cancer in a subject comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and/or analog thereof, and fluorouracil, TAS-102, oxaliplatin, venetoclax, or SN-38. In certain aspects, the fluorouracil, TAS-102, oxaliplatin, venetoclax, or SN-38 is administered to the subject in an effective amount.

Additionally, combination therapy is provided, wherein a subject having cancer is administered the combination therapy, wherein the combination therapy comprises administering to the subject having cancer an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and fluorouracil, TAS-102, oxaliplatin, venetoclax or SN-38.

In certain aspects, the antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR is administered in combination with fluorouracil, folinic acid and irinotecan (FOLFIRI) or folinic acid, fluorouracil and oxaliplatin (FOLFOX). In certain aspects, the subject has received prior therapy for the cancer. In certain aspects, the subject is administered the chemotherapy as a second-line therapy. In certain aspects, the subject is administered the chemotherapy as a third-line or subsequent therapy. In certain aspects, the subject suffers from colorectal cancer, particularly metastatic colorectal cancer (mCRC). In certain aspects, the treatment methods of the present disclosure further comprise administering to the subject 1500 mg of fetosemtamab every two weeks (Q2W). In certain embodiments, the subject has mCRC and is wild type for RAS and/or RAF (including Kirsten Rat Sarcoma (KRAS) and/or B-Rapidly Accelerated Fibrosarcoma (BRAF)). In certain embodiments, the genome of the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF.

In certain instances, the cancer is adenocarcinoma or squamous cell carcinoma. In certain instances, the cancer is head and neck cancer, stomach cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer. In certain instances, the cancer is colorectal cancer.

In certain aspects, the subject of the present disclosure is a mammalian subject, e.g., a human subject.

In certain instances, the subject has progressed following prior anticancer therapy, wherein the anticancer therapy is chemotherapy, targeted therapy, immunotherapy, or radiation therapy.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof exhibits enhanced ADCC. Furthermore, in certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is afucosylated.

In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof of the present disclosure are multispecific antibodies. In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof are bispecific antibodies. In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof are bispecific antibodies that bind at least EGFR. In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof comprise a second variable domain that does not bind EGFR. In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof monovalently bind EGFR. In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof comprise a second variable domain that binds LGR5.

In certain embodiments, the subject to be treated has not received prior anticancer therapy. In certain embodiments, the subject to be treated has not received prior chemotherapy, targeted therapy, immunotherapy, or radiation therapy. In certain embodiments, the subject to be treated has not received prior therapy with fluorouracil, oxaliplatin, FOLFOX, FOLFIRI, TAS-102, trastuzumab, pembrolizumab, nivolumab, cetuximab, or a combination thereof.

In certain embodiments, the antibody or functional portion, derivative and/or analog thereof is administered to the subject simultaneously, separately or sequentially with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides a pharmaceutical composition comprising an antibody or a functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure further provides a kit component comprising an antibody or functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 and instructions for use of the antibody or functional portion, derivative and/or analog thereof and instructions for use of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides the use of an antibody or a functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the manufacture of a medicament for treating cancer in a subject, wherein the treatment comprises administering simultaneously, separately or sequentially the antibody or a functional portion, derivative and/or analog thereof and the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure relates to the use of an antibody or a functional portion, derivative and/or analog thereof, comprising a variable domain that binds an extracellular portion of EGFR and further comprising a variable domain that binds an extracellular portion of LGR5, and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the manufacture of one or more medicaments for the treatment of cancer in a subject. In certain aspects, the antibody or a functional portion, derivative and/or analog thereof and the fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 are used in the manufacture of separate medicaments, e.g., two separate medicaments, one for said antibody or a functional portion, derivative and/or analog thereof and one for said fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. The medicament comprising the antibody, functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR may be contained in a holder that is separate from, i.e. not physically connected to, the holder containing the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 as a medicament.

In certain aspects, the antibody or functional portion, derivative and/or analog thereof, comprising a variable domain that binds the extracellular portion of EGFR and optionally comprising a variable domain that binds the extracellular portion of LGR5, is or comprises petosemtamab.

In certain aspects, the present disclosure provides the use of an antibody or functional portion, derivative and/or analog of the present disclosure comprising a variable domain capable of binding to the extracellular portion of EGFR in the manufacture of a medicament for enhancing the effectiveness of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for the treatment of cancer.

In certain aspects, the present disclosure provides the use of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 in the manufacture of a medicament for enhancing the effectiveness of an antibody or functional portion, derivative, and/or analog of the present disclosure comprising a variable domain capable of binding to the extracellular portion of EGFR for the treatment of cancer.

The present disclosure also provides a combination of an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR as described herein and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for use in the treatment of cancer in a subject in need thereof.

The present disclosure also provides combinations of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of cancer in a subject, instructions for use of said fluoropyrimidine, platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, as well as instructions for use of an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR in the treatment of said cancer in a subject.

The present disclosure also provides a combination of an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR, instructions for use of said antibody or a functional portion, derivative and/or analog thereof in the treatment of cancer in a subject, as well as instructions for use of said antibody or a functional portion, derivative and/or analog thereof in the treatment of cancer in a subject, with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides pharmaceutical compositions comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and instructions for use thereof with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of cancer.

In certain aspects, the present disclosure provides a pharmaceutical composition for the treatment of cancer, comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and a pharmaceutical composition for the treatment of said cancer, comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides a pharmaceutical composition for use in the treatment of cancer comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, wherein the pharmaceutical composition is administered in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 of the present disclosure.

In certain aspects, the present disclosure relates to a pharmaceutical composition for the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional portion, derivative and/or analog thereof, wherein the subject to be treated is administered a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, prior to or concurrently with administration of the antibody.

In certain aspects, the present disclosure relates to a pharmaceutical composition for treating cancer in a subject comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38, wherein the subject to be treated is administered an antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure prior to, concurrently with, or subsequent to administration of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor, or SN-38.

Accordingly, the present disclosure relates to a combination of medicaments for treating cancer in a subject, comprising administering to the subject a plurality of different medicaments for treating said cancer, wherein the treatment comprises simultaneous, sequential, or separate administration of the medicaments. In certain aspects, the medicament comprises an antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and the other different medicaments comprise a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure may be administered simultaneously, sequentially, or separately with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 of the present disclosure. Thus, the combination of the antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 encompasses simultaneous, sequential, or separate administration.

Accordingly, in certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure for use in a method of treating cancer, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, optionally wherein the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

Accordingly, in certain aspects, the present disclosure provides a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 and an antibody or functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, wherein optionally the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38.

Accordingly, in certain aspects, the present disclosure provides the use of an antibody or a functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the manufacture of a medicament for the treatment of cancer, wherein optionally the antibody or a functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38. In certain aspects, the antibody or a functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38.

In certain aspects, an antibody or a functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure is for use in the manufacture of a medicament for treating cancer, wherein a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 is for use in the manufacture of a medicament for treating said cancer, wherein optionally the antibody or a functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38. Optionally, the antibody or a functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

In certain aspects, the antibody or functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure is for use in the manufacture of a medicament for the treatment of cancer, whereby the treatment is combined with FOLFIRI, optionally wherein the antibody or functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the FOLFIRI. Optionally, the antibody or functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the FOLFIRI.

In certain aspects, the antibody or functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure is for use in the manufacture of a medicament for the treatment of cancer, whereby the treatment is combined with FOLFOX, optionally wherein the antibody or functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the FOLFOX. Optionally, the antibody or functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the FOLFOX.

Figure 1: Human LGR5 sequence; SEQ ID NO: 1.
Figure 2: Human EGFR sequence; SEQ ID NO: 2.
Figure 3: (a) Amino acid sequence of the heavy chain variable region (SEQ ID NOs: 3 to 15) that forms the variable domain that binds LGR5 and EGFR together with the common light chain variable region, e.g., the variable region of human kappa light chain IgVkV1-39/jk1 (SEQ ID NO: 107). The heavy chain CDRs (according to the KABAT numbering system) and framework regions are shown in Figure 3b. Figure 4: a) Amino acid sequence of the common light chain. b) Common light chain variable region (IgVk1-39/jk1). c) Light chain constant region. d) V-region IgVk1-39; e) CDR1, CDR2, and CDR3 of the common light chain according to the IMGT numbering.
Figure 5: IgG heavy chain for the production of bispecific molecules. a) CH1 domain DNA. b) Hinge domain. c) CH2 domain. d) CH3 domain containing mutations L351K and T366K (KK). e) CH3 domain containing mutations L351D and L368E (DE). Residue positions are according to EU numbering.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5 for use in treating cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In certain aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds an extracellular portion of EGFR and a variable domain that binds an extracellular portion of LGR5, and a fluoropyrimidine, a platinum-based chemotherapeutic, a BCL-2 inhibitor or SN-38.

Cancer type

In certain aspects, the cancer of the present disclosure is adenocarcinoma or squamous cell carcinoma.

In certain embodiments, the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal junction cancer, head and neck cancer, non-small cell lung cancer or colorectal cancer, particularly squamous cell carcinoma of the head and neck (SCCHN).

In certain instances, the cancer is stomach, esophageal, or gastroesophageal junction cancer. Gastric cancer (also called gastric cancer) is a cancer that arises from the gastric mucosa, specifically the mucus-producing glandular cells found within it. This cancer is also called adenocarcinoma, or in this case, gastric adenocarcinoma, which arises from the gastric mucosa. Specifically, this cancer is referred to as gastric adenocarcinoma or cancer arising from the gastric mucosa, which are used interchangeably herein. Esophageal cancer is cancer that arises from the esophagus. The two main subtypes are esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Gastroesophageal junction cancer (also called gastroesophageal junction adenocarcinoma) arises from the gastroesophageal junction.

Head and neck cancer, collectively known as head and neck cancer, typically originates from the squamous cells that line the moist mucosal surfaces of the head and neck, such as the mouth, nose, and throat. These squamous cell cancers, often referred to as squamous cell carcinoma of the head and neck, are treated in certain aspects of the present disclosure. Rarely, head and neck cancers can also arise in the salivary glands. Head and neck cancers, in particular, can arise in the oral cavity. This includes the lips, the anterior two-thirds of the tongue, the gums, the buccal and inner lip mucosa, the roof of the mouth, the hard palate, and a small area of the gums behind the wisdom teeth.

Accordingly, in certain aspects, the head and neck cancer is a squamous cell carcinoma, including nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal sinus cancer, oral cancer, oropharyngeal cancer, or salivary gland cancer. More specifically, the present disclosure relates to the treatment of cancers, including squamous cell head and neck cancer, located, for example, in the oropharynx, hypopharynx, larynx, oral cavity, or tongue. Furthermore, the head and neck cancer is particularly squamous cell carcinoma of unknown primary (also known in the art as cancer of unknown primary or CUP).

In certain instances, the cancer is non-small cell lung cancer.

In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is metastatic colorectal cancer (mCRC). In certain embodiments, the subject has been diagnosed with adenocarcinoma of the colon or rectum. In certain embodiments, the cancer has been previously treated with, for example, standard first-line therapy appropriate for CRC or mCRC. In certain embodiments, the subject or the cancer has been treated with, and is receiving, second-line therapy using an antibody or functional portion, derivative, and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5 in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38.

In certain aspects, the subject or cancer has been or is treated with a second-line therapy comprising an antibody or functional portion thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5, or a derivative and/or analog thereof, in combination with a fluoropyrimidine, a platinum-based chemotherapy agent, a BCL-2 inhibitor, or SN-38 as a third-line therapy or subsequently. In certain aspects, the prior therapy comprises an appropriate standard therapy for CRC or mCRC. In certain aspects, the standard therapy comprises or is an appropriate chemotherapy for CRC or mCRC. In certain aspects, the prior therapy comprises or is an appropriate chemotherapy for CRC or mCRC. In certain aspects, the prior therapy comprises or is an appropriate fluoropyrimidine-oxaliplatin-based chemotherapy with or without bevacizumab or an appropriate fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab for CRC or mCRC.

In certain embodiments, the subject to be treated suffers from CRC and is wild type for RAS. In certain embodiments, the subject to be treated suffers from CRC and is wild type for RAF. RAS comprises Kirsten rat sarcoma (KRAS) and RAF comprises B-rapidly accelerated fibrosarcoma (BRAF). In certain embodiments, the genome of the CRC is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the genome of a sample obtained from the CRC is wild type for RAS and/or RAF, including KRAS and/or BRAF.

In certain embodiments, the subject to be treated has mCRC and is wild type for RAS. In certain embodiments, the subject to be treated has mCRC and is wild type for RAF. RAS includes Kirsten rat sarcoma (KRAS), and RAF includes B-rapidly accelerated fibrosarcoma (BRAF). In certain embodiments, the genome of the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the genome of a sample obtained from the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the subject has previously been diagnosed with unresectable or metastatic colon or rectal adenocarcinoma, histologically or cytologically confirmed. In certain embodiments, the subject is RAS and/or RAF wild-type, as determined using next-generation sequencing (NGS) of tumor tissue (primary or metastatic). In certain embodiments, the subject has not received prior therapy with an anti-EGFR therapy. In other words, the subject is naive to prior anti-EGFR therapy. In certain embodiments, the subject has radiologically confirmed disease that developed during or within 6 months of the prior (first-line) chemotherapy.

In certain embodiments, the cancer is a cancer that expresses EGFR. In certain embodiments, the cancer is a cancer that expresses LGR5. In certain embodiments, the cancer is a cancer that expresses EGFR and LGR5.

In certain embodiments, the cancer is gastric cancer that expresses EGFR. In certain embodiments, the cancer is esophageal cancer that expresses EGFR. In certain embodiments, the cancer is gastroesophageal junction cancer that expresses EGFR. In certain embodiments, the cancer is head and neck cancer that expresses EGFR. In certain embodiments, the cancer is non-small cell lung cancer that expresses EGFR. In certain embodiments, the cancer is colorectal cancer that expresses EGFR. In certain embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN) that expresses EGFR.

As used herein, if a cancer cell comprises a cell that expresses EGFR, the cancer cell expresses EGFR. Cells that express EGFR contain detectable levels of RNA encoding EGFR. In certain embodiments, EGFR expression is determined by ISH. As used herein, if a cancer cell comprises a cell that expresses LGR5, the cancer cell expresses LGR5. Cells that express LGR5 contain detectable levels of RNA encoding LGR5.

In certain embodiments, EGFR protein expression is detected by IHC. In certain embodiments, EGFR expression is determined by IHC using a commercially available EGFR detection kit, such as the EGFR pharmDx™ kit for the Dako autostainer (Agilent), following the manufacturer's recommendations, or using a commercially available IHC EGFR detection kit based on EGFR clone 113, which binds to the EGFR extracellular domain (Leica, https://shop.leicabiosystems.com/us/ihc-ish/ihc-primary-antibodies/pid-epidermal-growth-factor-receptor). Alternatively, EGFR expression is determined using Novocastra™ liquid mouse monoclonal antibody epidermal growth factor receptor (product code: NCL-L-EGFR, Epidermal Growth Factor Receptor - IHC Primary Antibodies by leicabiosystems.com), based on clone EGFR.113.

Briefly, the commercially available EGFR pharmDx™ IHC kit system contains the reagents necessary to complete the IHC staining procedure of routinely fixed, paraffin-embedded specimens. After incubation with a primary non-Her2, Her3, and Her4 cross-reactive monoclonal antibody (clone 2-18C9) against the human EGFR protein, the kit utilizes a ready-to-use visualization reagent based on dextran technology. This reagent consists of a secondary goat anti-mouse antibody molecule and a horseradish peroxidase molecule linked to a common dextran polymer backbone. Subsequent enzymatic conversion of the added chromogen results in the formation of a visible reaction product at the antigen site. Results are routinely evaluated using light microscopy. Control slides containing two formalin-fixed, paraffin-embedded human cell lines with staining intensity scores of 2+ and 0 are provided for quality control of the kit reagent performance.

Staining intensity is scored as follows: 3+ (strong staining): visible at low magnification, such as a 5x objective, and can be identified at higher magnifications if necessary; 2+ (moderate staining): visible at intermediate magnification, such as a 10x or 20x objective; 1+ (weak staining): reliably identified only at high magnification, such as a 40x objective; 0 (no staining): staining not visible at high magnification.

In certain instances, EGFR expression is determined using immunohistochemistry (IHC), and the cancer is IHC positive for EGFR. In certain instances, the cancer is characterized by an EGFR IHC score of 2+ or 3+. In certain instances, the cancer is characterized by an H-score for EGFR of greater than 50, greater than 80, or greater than 200 to less than 300.

In certain embodiments, EGFR expression is determined using immunohistochemistry (IHC) and then assigned an H-score for EGFR using a scale of 0 to 300. In certain embodiments, the cancer of the present disclosure is characterized by an H-score for EGFR of greater than 200 on a scale of 0 to 300. Thus, in certain embodiments, the EGFR H-score is between 200 and 300. In certain embodiments, the cancer of the present disclosure is characterized by an H-score for EGFR of greater than 50 on a scale of 0 to 300. Thus, in certain embodiments, the EGFR H-score is between 50 and 300. In certain embodiments, the cancer of the present disclosure is characterized by an H-score for EGFR of greater than 80 on a scale of 0 to 300. Thus, in certain embodiments, the EGFR H-score is between 80 and 300.

In the present disclosure, the determination of the H-score for assigning EGFR expression status involves a first step of establishing a membrane staining intensity (score of 0, 1+, 2+, or 3+), which is determined for each cell in a predefined region as described herein. Thereafter, the percentage of cells at each staining intensity level is calculated, and finally an H-score is assigned using the following formula to generate an H-score for EGFR ranging from 0 to 300: [1 × (% of cells with 1+ staining) + 2 × (% of cells with 2+ staining) + 3 × (% of cells with 3+ staining)]. Consequently, the H-score assigns greater relative weight to higher staining intensities or amounts in a given tumor sample.

Optionally, treatment with the antibody or functional portion, derivative and/or analog thereof comprises (or in certain aspects is preceded by) diagnosing the EGFR status of the subject. In certain aspects, a subject is selected for treatment having an IHC score of 3+ or a cancer characterized by an H-score for EGFR of greater than 200 on a scale of 0 to 300. In certain aspects, treatment of the subject is preceded by diagnosing the subject by an H-score for EGFR of greater than 50 on a scale of 0 to 300. In certain aspects, treatment of the subject is preceded by diagnosing the subject by an H-score for EGFR of greater than 80 on a scale of 0 to 300. In certain aspects, treatment of the subject is preceded by diagnosing the subject by an H-score for EGFR of greater than 200 on a scale of 0 to 300. After diagnosis, subjects with scores falling within a selected range (i.e., 50 to 300 points, 80 to 300 points, or 200 to 300 points) are selected for treatment according to the present disclosure.

In certain embodiments, the cancer expresses LGR5. As used herein, a cancer cell expresses LGR5 if it comprises a cell expressing LGR5. Cells expressing LGR5 contain detectable levels of RNA encoding LGR5.

Expression can often be detected by incubating cells with antibodies that bind to LGR5. However, some cells do not express the protein at sufficiently high levels for such antibody testing. In these cases, detection of mRNA or other forms of nucleic acid sequences is preferred. In certain embodiments, LGR5 is detected through mRNA expression. In certain embodiments, LGR5 detection is performed by RNA sequencing. In certain embodiments, LGR5 detection is performed by tissue microarray (TMA). In certain embodiments, LGR5 expression is determined using in situ hybridization (ISH). Therefore, preferably, the cancer is ISH-positive for LGR5. ISH-positivity preferably means that expression is characterized by an H-score of 1 or greater.

Techniques for detection and scoring based on TMA, ISH, and IHC are well known to those skilled in the art and are generally commercially available as standard kits. Quantification of mRNA levels using ISH and expression based on H-scores, such as for LGR5, can be performed using commercially available kits, such as the RNAscope® kit from Advanced Cell Diagnostics (Hayward, CA), using a staining platform such as the BondRx platform (Leica, Wetzlar, Germany). ISH H-scores for LGR5 quantification typically range from 0 to 400. Alternatively, LGR5 expression is determined by RNA sequencing (RNAseq).

In certain aspects, the cancer expresses LGR5 at a sufficient level for the antibody to bind to the LGR5 protein, e.g., the antibody comprises a VH chain of an LGR5-binding variable domain comprising the amino acid sequence of the VH chain of MF5816 as illustrated in FIG. 3 or an alternative variable domain that binds LGR5 described herein. In certain aspects, the cancer expresses EGFR at a sufficient level for the antibody to bind to the EGFR protein, e.g., the antibody comprises a VH chain of an EGFR-binding variable domain comprising the amino acid sequence of the VH chain of MF3755 as illustrated in FIG. 3 or an alternative variable domain that binds EGFR described herein.

Mutations and Models

Cancers, such as gastric, esophageal, gastroesophageal junction, head and neck, non-small cell lung, or colorectal cancer, may be associated with the presence of mutations. These mutations include mutations in known oncogenes, such as PIK3CA, RAS, KRAS, HRAS, MAP2K1, RAF, BRAF, and NOTCH1. Oncogenic mutations are generally described as activating mutations or mutations that introduce new functions. Another type of cancer mutation involves tumor suppressor genes, such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are typically inactivating.

In certain embodiments, the cancer and/or the subject having the cancer has a mutation in one or more WNT, RTK/RAS, TP53 and/or TGF-β signaling pathway genes.

In certain embodiments, the cancer is colorectal cancer and is wild type for RAS and/or RAF (including Kirsten rat sarcoma (KRAS) and/or B-rapidly accelerating fibrosarcoma (BRAF)). In certain embodiments, the genome of the colorectal cancer is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the cancer is mCRC and is wild type for RAS and/or RAF (including Kirsten rat sarcoma (KRAS) and/or B-rapidly accelerating fibrosarcoma (BRAF)). In certain embodiments, the genome of the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF.

In certain embodiments, for subjects with mCRC, a screening step is included to detect (and optionally to exclude subjects with) somatic mutations in RAS and RAF family genes (i.e., KRAS, NRAS, HRAS, BRAF, ARAF, RAF1). In certain embodiments, a screening step is included to detect mutations associated with resistance to antibody-based EGFR inhibition or other oncogenic factors, such as EGFR ectodomain or ERBB2/HER2 amplification.

In certain embodiments, the cancer has an oncogenic mutation in one or more genes associated with colorectal cancer. In certain embodiments, the cancer has an oncogenic mutation in a gene selected from APC, FBXW7, KRAS, PIK3CA, SOX9, TP53, SMAD2, SMAD3, SMAD4, NRAS, or TCF7L2 and a corresponding encoded protein. In certain embodiments, the cancer has a mutation in a gene selected from APC, FBXW7, KRAS, PIK3CA, SOX9, TP53, BRAF, SMAD2, SMAD3, SMAD4, NRAS, or TCF7L2 and a corresponding encoded protein.

In certain embodiments, the cancer has a mutation in the gene encoding APC. In certain embodiments, the mutation is a missense mutation, a nonsense mutation, a frameshift mutation, an insertion, a deletion, or a copy number change. In certain embodiments, the mutation is a monoallelic loss-of-function, biallelic loss-of-function, or loss-of-heterozygosity mutation. In certain embodiments, the mutation is a biallelic loss-of-function mutation. In certain embodiments, APC contains a monoallelic loss-of-function missense mutation R653K in the protein structure, which results in an R>K amino acid change. In certain embodiments, this mutation is a heterozygous loss-of-function mutation, which results in a copy number change. In certain embodiments, APC contains a biallelic loss-of-function mutation at position 4704, which results in a frameshift at position P1442fs*31 of the protein. In certain embodiments, APC comprises a biallelic loss-of-function mutation at positions 5040-5041, which causes a frameshift at position T1556fs*3 of the protein. In certain embodiments, APC comprises a mutation at positions 4565-4566, which causes a frameshift at position F1396fs*1 of the protein.

Any mutations mentioned herein follow standard nomenclature understood by those of skill in the art. As used herein with respect to mutations, it will be apparent to those of skill in the art that an asterisk (*) indicates the presence of a stop codon and thus translation termination. Also, frameshift mutations are described herein in the format X ₁ fs*X ₂ , where X ₁ represents the first affected amino acid in the protein and *X ₂ represents the position of the translation termination codon in the new reading frame.

In certain instances, cancers harbor mutations in the gene encoding FBXW7. In certain instances, the mutations are missense mutations or copy number altering mutations. In certain instances, the mutations are monoallelic loss-of-function mutations or loss-of-heterozygosity mutations.

In certain instances, the cancer harbors a mutation in the gene encoding KRAS. In certain instances, the mutation is a missense mutation. In certain instances, the mutation is a gain-of-function mutation. In certain instances, KRAS contains a G12V mutation in its protein structure, resulting in a G>V amino acid change. In certain instances, KRAS contains a Q61H mutation in its protein structure, resulting in a Q>H amino acid change. In certain instances, KRAS contains a G12S mutation in its protein structure, resulting in a G>S amino acid change.

In certain cases, the cancer harbors a mutation in the gene encoding PIK3CA. In certain cases, the mutation is a missense mutation. In certain cases, the mutation is a gain-of-function mutation. In certain cases, PIK3CA contains a gain-of-function missense mutation, E545K, in its protein structure, resulting in an amino acid change from E to K.

In certain embodiments, the cancer has a mutation in the gene encoding SOX9. In certain embodiments, the mutation is a nonsense mutation or a frameshift mutation. In certain embodiments, the mutation is a monoallelic loss-of-function mutation. In certain embodiments, SOX9 contains a monoallelic loss-of-function nonsense mutation Q412* within its protein structure, which results in an amino acid change Q>*. In certain embodiments, SOX9 contains a monoallelic loss-of-function mutation at positions 1898 to 1899, which results in a frameshift at position *510fs*68 of the protein.

In certain embodiments, the cancer has a mutation in the gene encoding TP53. In certain embodiments, the mutation is a missense mutation or a nonsense mutation. In certain embodiments, the mutation is a monoallelic loss-of-function mutation or a biallelic loss-of-function mutation. In certain embodiments, TP53 comprises a missense mutation N239D in its protein structure, which results in an N>D amino acid change. In certain embodiments, TP53 comprises a biallelic loss-of-function nonsense mutation Y126* in its protein structure, which results in an Y>* amino acid change. In certain embodiments, TP53 comprises a monoallelic loss-of-function missense mutation R282W in its protein structure, which results in an R>W amino acid change. In certain embodiments, TP53 comprises a biallelic loss-of-function missense mutation M237I in its protein structure, which results in an M>I amino acid change.

In certain cases, cancers harbor mutations in the gene encoding SMAD4. In certain cases, the mutations are missense or loss-of-heterozygosity mutations. In certain cases, SMAD4 contains a missense mutation, G510V, within its protein structure, resulting in a G>V amino acid change.

In certain instances, the cancer harbors mutations in genes selected from SMAD2, SMAD3, SMAD4, NRAS, or TCF7L2. In certain instances, the mutations are loss-of-heterozygosity mutations. In certain instances, the mutations result in copy number changes.

In certain aspects, the present disclosure relates to a method of treating cancer in a subject having a mutation in one or more genes selected from APC, SOX9, KRAS, NRAS, SMAD2, or SMAD4. In certain aspects, the cancer has a mutation in the genes APC, SOX9, KRAS, NRAS, SMAD2, and SMAD4. In certain aspects, the mutation in the gene encoding APC is a monoallelic loss-of-function missense mutation R653K in the protein structure, which results in an R>K amino acid change. In certain aspects, the mutation in the gene encoding APC is a mutation at positions 4565-4566, which results in a frameshift at position F1396fs*1 of the protein. In certain aspects, the mutation in the gene encoding SOX9 is a monoallelic loss-of-function nonsense mutation Q412* in the protein structure thereof, which results in an Q>* amino acid change. In certain instances, mutations in the gene encoding KRAS are G12V mutations within its protein structure, resulting in a G>V amino acid change. In certain instances, mutations in the genes NRAS, SMAD2, and SMAD4 are loss-of-heterozygosity mutations, resulting in copy number changes.

In certain aspects, the present disclosure relates to a method of treating cancer in a subject having a mutation in APC.

In certain aspects, the present disclosure relates to a method of treating cancer in a subject having a mutation in one or more genes selected from TP53, APC, NRAS, SMAD2, SMAD4, or FBXW7. In certain aspects, the cancer has a mutation in the genes TP53, APC, NRAS, SMAD2, SMAD4, and FBXW7. In certain aspects, the mutation in the genes encoding APC, NRAS, SMAD2, SMAD4, and FBXW7 is a loss-of-heterozygosity mutation, which results in a copy number change. In certain aspects, the mutation in the gene encoding TP53 is a missense mutation N239D in its protein structure, which results in an N>D amino acid change. In certain aspects, the mutation in the gene encoding TP53 is a biallelic loss-of-function nonsense mutation Y126* in its protein structure, which results in an Y>* amino acid change.

In certain aspects, the present disclosure relates to a method of treating cancer in a subject having a mutation in one or more genes selected from TP53, APC, KRAS, or PIK3CA. In certain aspects, the cancer has mutations in the genes TP53, APC, KRAS, and PIK3CA. In certain aspects, the mutation in the gene encoding APC is a biallelic loss-of-function mutation at position 4704, which causes a frameshift in its protein at position P1442fs*31. In certain aspects, the mutation in the gene encoding KRAS is a mutation G12S in its protein structure, which results in a G>V amino acid change. In certain aspects, the mutation in the gene encoding PIK3CA is a gain-of-function missense mutation E545K in its protein structure, which results in an E>K amino acid change. In a specific aspect, the mutation in the gene encoding TP53 is a monoallelic loss-of-function missense mutation R282W within its protein structure, which results in an R>W amino acid change.

In certain aspects, the present disclosure relates to a method of treating cancer in a subject having a mutation in one or more genes selected from TP53, APC, SOX9, KRAS, PIK3CA, TCF7L2, NRAS, SMAD2, SMAD3, or SMAD4. In certain aspects, the cancer has a mutation in the genes TP53, APC, SOX9, KRAS, PIK3CA, TCF7L2, NRAS, SMAD2, SMAD3, and SMAD4. In certain aspects, the mutations in the genes encoding APC, TCF7L2, NRAS, SMAD2, SMAD4, and TP53 are heterozygous loss-of-function mutations, which result in copy number changes. In certain aspects, the mutations in the gene encoding APC are biallelic loss-of-function mutations at positions 5040-5041, which result in a frameshift at position T1556fs*3 of its protein. In certain aspects, the mutation in the gene encoding SOX9 is a monoallelic loss-of-function mutation at positions 1898-1899, which causes a frameshift at position *510fs*68 in its protein. In certain aspects, the mutation in the gene encoding KRAS is a Q61H mutation in its protein structure, which results in a Q>H amino acid change. In certain aspects, the mutation in the gene encoding PIK3Ca is a E545K gain-of-function missense mutation in its protein structure, which results in an E>K amino acid change. In certain aspects, the mutation in the gene encoding SMAD4 is a G510V missense mutation in its protein structure, which results in a G>V amino acid change. In certain aspects, the mutation in the gene encoding TP53 is a biallelic loss-of-function missense mutation M237I in its protein structure, which results in an M>I amino acid change.

Standard therapeutic molecules and their administration in the present disclosure

Fluoropyrimidines

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering a fluoropyrimidine. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of a fluoropyrimidine.

Fluoropyrimidines are antimetabolites that inhibit thymidylate synthetase and reduce the production of the pyrimidine thymidine. Without wishing to be bound by theory, it is believed that fluoropyrimidines interfere with DNA synthesis and, to a lesser extent, RNA synthesis, leading to apoptosis in rapidly growing cells.

Examples of fluoropyrimidines suitable for humans include capecitabine, carmophor (HCFU), doxifluridine, 5-fluorouracil (5-FU), and tegafur. In certain aspects, "fluoropyrimidine" as used herein includes, but is not limited to, capecitabine, carmophor (HCFU), doxifluridine, 5-fluorouracil (5-FU), and tegafur.

In certain embodiments, the fluoropyrimidine is fluorouracil.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering fluorouracil. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and fluorouracil.

Fluorouracil is a fluoropyrimidine with the chemical formula 5-fluoro-2,4(1H,3H)-pyrimidinedione. Fluorouracil is converted to three major active metabolites: 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), 5-fluorouridine-5'-triphosphate (FUTP), and 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP). These metabolites have several effects, including inhibition of thymidylate synthase by FdUMP, incorporation of FUTP into RNA, and incorporation of FdUTP into DNA.

Fluorouracil is known by various names, the most common being 5-fluorouracil, 5-fluoropyrimidine-2,4-dione, 5-FU, and 5-fluorouracil. Fluorouracil is sold under the brand name Adrucil. The IUPAC name for fluorouracil is 5-fluoropyrimidine-2,4(1H,3H)-dione. It is used intravenously to treat colorectal, esophageal, stomach, pancreatic, breast, and cervical cancers. It is also used as a cream to treat actinic keratoses, basal cell carcinoma, and skin warts.

Fluorouracil and other human fluoropyrimidines have been used clinically for a long time, and appropriate regimens and dosage information are readily available to those skilled in the art. Fluorouracil can be administered intravenously, e.g., by bolus, infusion, or continuous infusion, for up to several days. Fluorouracil can be administered monotherapy, in combination with leucovorin alone, or in combination with leucovorin and oxaliplatin or irinotecan. Fluorouracil can also be administered as a component of cyclophosphamide-based multidrug regimens or as a component of platinum-containing multidrug chemotherapy regimens.

In certain embodiments, the dosage regimen of fluorouracil comprises intravenous administration of fluorouracil in the range of 200 to 3000 mg/m ² .

In certain embodiments, the dosing regimen of fluorouracil comprises administering 400 mg/m ² intravenously as a bolus on day 1, followed by 2400 mg/m ² to 3000 mg/m ² intravenously as a continuous infusion over 46 hours every two weeks, in combination with leucovorin alone or in combination with leucovorin and oxaliplatin or irinotecan.

In a specific aspect, the fluorouracil dosing regimen comprises administering fluorouracil as an intravenous bolus at 500 mg/m ² in combination with leucovorin on days 1, 8, 15, 22, 29, and 36 of an 8-week cycle.

In certain embodiments, the fluorouracil dosing regimen comprises intravenous administration of 500 mg/m ² to 600 mg/m ² of fluorouracil on days 1 and 8 every 28 days for 6 cycles as a component of a cyclophosphamide-based multidrug regimen.

In certain embodiments, the fluorouracil dosing regimen comprises administering fluorouracil intravenously as a continuous infusion over 24 hours at a dose of 200 mg/m ² to 1000 mg/m ² as a component of a platinum-containing multi-agent chemotherapy regimen.

In certain embodiments, the dosing regimen of fluorouracil comprises administering fluorouracil in combination with leucovorin or as a component of a multi-agent chemotherapy regimen including leucovorin as an intravenous bolus of 400 mg/m ² on day 1, followed by 2400 mg/m ² as a continuous infusion intravenously over 46 hours every two weeks thereafter.

TAS-102

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering a fluoropyrimidine. In certain embodiments, the method of treating cancer in a subject further comprises administering an effective amount of a fluoropyrimidine.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment that further comprises administering a thymidine phosphorylase inhibitor. In certain embodiments, the method of treating cancer in a subject further comprises administering an effective amount of thymidine phosphorylase.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering a fluoropyrimidine and a thymidine phosphorylase inhibitor. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of a fluoropyrimidine and an effective amount of thymidine phosphorylase.

In certain embodiments, the fluoropyrimidine is trifluridine. In certain embodiments, the thymidine phosphorylase inhibitor is tipiracil hydrochloride.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering trifluridine and tipiracil hydrochloride. In certain aspects, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab, trifluridine, and tipiracil hydrochloride.

In a specific aspect, the combination of trifluorothymidine and tipiracil in a molar ratio of 1:0.5 is TAS-102.

Trifluridine, an antineoplastic thymidine-based nucleoside analogue, is chemically described as 2'-deoxy-5-(trifluoromethyl)uridine. Tipiracil hydrochloride, a thymidine phosphorylase inhibitor, is chemically described as 5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]pyrimidine-2,4-(1H,3H)-dione hydrochloride or 2,4(1H,3H)-pyrimidinedione, 5-chloro-6-[(2-imino-1-pyrrolidinyl)methyl]-, hydrochloride (1:1). TAS-102 contains trifluridine and tipiracil in a molar ratio of 1:0.5 (1:00471 by weight). Without wishing to be bound by theory, the inclusion of tipiracil is thought to increase trifluridine exposure by inhibiting thymidine phosphorylase-mediated trifluridine metabolism. After trifluridine is absorbed into cancer cells, it is incorporated into their DNA, interfering with DNA synthesis and inhibiting cell proliferation.

TAS-102 is known by several synonyms, the most common of which are Lonsurf, TAS 102, TAS-102 (trifluridine/tipiracil HCl), 4-hydroxy-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-(trifluoromethyl)pyrimidin-2(1H)-one compound with 5-chloro-6-((2-iminopyrrolidin-1-yl)methyl)pyrimidine-2,4-diol (1:1) hydrochloride, Viroptic mixture with 5-CIMU, trifluridine-tipiracil hydrochloride mixture, tipiracil/trifluridine, and EX-A1755. TAS-102 is sold under the brand name Lonsurf.

TAS-102 has been used clinically for a long time, and appropriate regimen and dosing information is available to those skilled in the art. TAS-102 is indicated as a monotherapy for the treatment of adult patients with metastatic colorectal cancer (CRC) who have been previously treated with available therapies, including fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, anti-VEGF agents, and anti-EGFR agents, or who are not considered treatment candidates. TAS-102 is also indicated as a monotherapy for the treatment of adult patients with metastatic gastric cancer, including gastroesophageal junction adenocarcinoma, who have been previously treated with at least two prior systemic therapies for advanced disease.

In certain embodiments, the dosing regimen of TAS-102 comprises trifluorothymidine and tipiracil in a molar ratio of 1:0.5 at 35 to 80 mg/m ² twice daily.

In a specific aspect, the dosing regimen of TAS-102 comprises trifluorothymidine and tipiracil in a molar ratio of 1:0.5 administered orally twice daily on days 1 to 5 and days 8 to 12 of a 28-day cycle, starting at a dose of 35 mg/m ² /dose up to a maximum of 80 mg/dose, and continued until benefit is observed or unacceptable toxicity occurs.

Oxaliplatin

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment that further comprises a platinum-based chemotherapeutic agent. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of a platinum-based chemotherapeutic agent.

Platinum-based chemotherapeutics are antineoplastic drugs widely used in cancer treatment. They are platinum-based coordination complexes. Without wishing to be bound by theory, platinum-based chemotherapeutics are believed to induce DNA cross-linking, which inhibits DNA repair and/or DNA synthesis.

Examples of platinum-based chemotherapeutic agents suitable for humans include cisplatin, oxaliplatin, carboplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In certain aspects, "platinum-based chemotherapeutic agents" as used herein include, but are not limited to, cisplatin, oxaliplatin, carboplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. Cisplatin, oxaliplatin, nedaplatin, and carboplatin are approved and widely used for the treatment of various cancer indications, while triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin are currently in clinical trials.

In certain instances, the platinum-based chemotherapy agent is cisplatin, oxaliplatin, or carboplatin. In certain instances, the platinum-based chemotherapy agent is oxaliplatin.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering oxaliplatin. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and oxaliplatin.

Oxaliplatin has the molecular formula C ₈ H ₁₄ N ₂ O ₄ Pt and the chemical name cis-[(1R,2R)-1,2-cyclohexanediamine-N,N'][oxalato(2-)-O,O'] platinum. Oxaliplatin is an organoplatinum complex in which the platinum atom is complexed with 1,2-diaminocyclohexane (DACH) and an oxalate ligand as a leaving group. Oxaliplatin is nonenzymatically converted to active derivatives in physiological solutions through displacement of the labile oxalate ligand. These derivatives form inter- and intrastrand DNA cross-links, inhibiting DNA replication and transcription.

Oxaliplatin is known by various synonyms, the most common of which are diaminocyclohexane oxalatoplatinum, L-OHP, oxalatoplatin, oxalatoplatinum, oxaliplatin, Dacplat, Eloxatin, or Elplat. The brand name for oxaliplatin is Eloxatin. Oxaliplatin is used in combination with fluorouracil and folinic acid for the adjuvant treatment of stage III (Duke C) colon cancer following complete resection of the primary tumor and metastatic colorectal cancer.

Oxaliplatin and other platinum-based chemotherapeutics have been used clinically for a long time, and appropriate regimens and dosing information are readily available to those skilled in the art. In certain instances, oxaliplatin dosing regimens include administering oxaliplatin at a dose ranging from about 65 to 160 mg/m ² .

In a specific aspect, the oxaliplatin dosing regimen includes administering oxaliplatin at a dose of 85 mg/m ² every two weeks.

In certain instances, oxaliplatin is administered every two weeks in combination with fluorouracil and leucovorin.

In a specific embodiment, oxaliplatin is administered as an intravenous infusion at 85 mg/m ² over 120 minutes in combination with leucovorin at 200 mg/m ² , followed by fluorouracil as an intravenous bolus at 400 mg/m ² over 2 to 4 minutes, followed by fluorouracil as a continuous infusion at 600 mg/m ² over 22 hours.

Venetoclax

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment that further comprises a BCL-2 inhibitor. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of a BCL-2 inhibitor.

B-cell lymphoma 2 (BCL-2) is part of the apoptosis regulator family. BCL-2 is a pro-survival protein that, when overexpressed, protects cells from apoptosis. BCL-2 is overexpressed in many cancers and plays a crucial role in the negative regulation of apoptosis. Its expression is associated with increased drug resistance and tumor cell survival.

BH3 mimetics comprise a class of BCL-2 inhibitors that have shown promising results in several hematological malignancies, both as single agents and in combination with other anticancer drugs. Without wishing to be bound by theory, BH3 mimetics are believed to inhibit BCL-2 activity and restore apoptosis in tumor cells. Suitable BCL-2 inhibitors in humans include ABT-737, navitoclax (ABT-263), and venetoclax.

In certain aspects, “BCL-2 inhibitors” as used herein include, but are not limited to, ABT-737, navitoclax (ABT-263), or venetoclax.

In certain instances, the BLC-2 inhibitor is venetoclax.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering venetoclax. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and venetoclax.

Venetoclax is a potent and selective inhibitor of the anti-apoptotic protein BCL-2. Venetoclax directly binds to the BH3-binding groove of BCL-2, displacing BH3-containing pro-apoptotic proteins such as BIM, thereby inducing mitochondrial outer membrane permeabilization (MOMP), caspase activation, and programmed cell death. In nonclinical studies, venetoclax exhibited cytotoxic activity in tumor cells overexpressing BCL-2.

Venetoclax is known by various synonyms, the most common of which are Venclexta, Venclyxto, ABT-199, GDC-0199, ABT199, ABT 199, and RG7601. Venclexta is used to treat patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). It is also used in combination with azacitidine, decitabine, or low-dose cytarabine to treat newly diagnosed acute myeloid leukemia (AML) in adults who are not suitable for intensive chemotherapy.

Venetoclax has been used clinically for a long time, and information on appropriate therapy and dosage is available to those skilled in the art.

In certain instances, venetoclax is administered daily at a dose of 20 to 1200 mg per day.

In certain instances, venetoclax is administered using an escalating or stepwise dosing regimen. In certain instances, the initial dose of venetoclax is gradually increased in incremental doses until the maximum dose is reached. In certain instances, the incremental doses are administered at regular intervals.

In a specific pattern, the initial dose of venetoclax is 20 mg in week 1, 50 mg in week 2, 100 mg in week 3, 200 mg in week 4, and 400 mg in week 5.

In certain embodiments, venetoclax is administered at a dose of 400 mg once daily according to an escalating dosing schedule as described herein. In certain embodiments, venetoclax is administered at a dose of 400 mg once daily until disease progression or unacceptable toxicity is observed.

SN-38

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering SN-38. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of SN-38.

SN-38 is 7-ethyl-10-hydroxy-camptothecin, a compound that inhibits the activity of DNA topoisomerase 1. It is the biologically active, water-insoluble metabolite of irinotecan (CPT-11). Without wishing to be bound by theory, it is believed that irinotecan is converted to SN-38 by carboxylesterase in the liver and tumors. SN-38 has been demonstrated to exhibit up to 1,000 times more potent cytotoxic activity than irinotecan against various cancer cells in vitro. However, the metabolic conversion rate is very low, with less than 10% of the original volume of irinotecan being metabolized to SN-38; the conversion of irinotecan to SN-38 varies depending on genetic variability in carboxylesterase activity between individuals. Direct administration of SN-38 itself for clinical cancer treatment eliminates the need for conversion. SN-38 is known to be equivalent to irinotecan in terms of efficacy and toxicity (Nakajima TE, et al. Int J Cancer. 2008 May 1; 122(9): 2148-53). Those skilled in the art will understand that the term "administering SN-38 to a subject" herein does not encompass administering irinotecan to the subject and then converting it to SN-38. Those skilled in the art will also understand that any term referring to "administering SN-38 to a subject" herein also encompasses administering a pharmaceutical composition comprising SN-38 to the subject.

SN-38 is known by various synonyms, the most common of which are 7-ethyl-10-hydroxycamptothecin, SN-38, SN38, SN-38 lactone, NK012, NK-012, 7-ethyl-10-hydroxy-20(S)-camptothecin, and 10-hydroxy-7-ethylcamptothecin. The IUPAC name is (4S)-4,11-diethyl-4,9-dihydroxy-1,4-dihydro-3H,14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione. LE-SN-38 is a novel liposome-based formulation comprising liposomes with a uniform size distribution (<200 nm). The drug entrapment efficiency of this formulation is greater than 95%.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering SN-38. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and SN-38.

In certain embodiments, SN-38 is administered encapsulated or conjugated to a suitable carrier, including a soluble polymer, liposome, micelle, antibody, peptide, polymer-drug conjugate, nanoparticle, or polymer implant.

FOLFOX

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering oxaliplatin, folinic acid (leucovorin), and fluorouracil. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of oxaliplatin, folinic acid, and fluorouracil.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering FOLFOX. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and FOLFOX.

In certain instances, the combination of oxaliplatin, folinic acid, and fluorouracil is called FOLFOX therapy. FOLFOX is known by various synonyms, the most common being the FOLFOX protocol, FOLFOX regimen, FOLFOX-4, FOLFOX-6, FOLFOX-7, and leucovorin calcium plus oxaliplatin with fluorouracil. Folinic acid is also known by the synonyms leucovorin, FA, and folinate calcium.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering oxaliplatin, folinic acid, and fluorouracil. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab, oxaliplatin, folinic acid, and fluorouracil.

In certain embodiments, the antibody or functional portion, derivative and/or analog thereof is used in a treatment additionally comprising FOLFOX (fluorouracil, leucovorin and oxaliplatin). In certain embodiments, the subject being treated has mCRC and is wild type for RAS and/or RAF (including Kirsten rat sarcoma (KRAS) and/or B-rapidly accelerated fibrosarcoma (BRAF)). In certain embodiments, the genome of the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the subject to be administered FOLFOX has received only one prior anticancer treatment, e.g., chemotherapy for metastatic disease, e.g., first-line fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab.

In certain embodiments, the FOLFOX dosing regimen comprises intravenous administration of oxaliplatin together with folinic acid followed by intravenous administration of fluorouracil.

In certain embodiments, the FOLFOX dosing regimen comprises intravenous administration of oxaliplatin 50 to 200 mg/m ² together with folinic acid 200 to 600 mg/m ² followed by intravenous administration of fluorouracil 1200 to 3600 mg/m ² .

In a specific aspect, the FOLFOX dosing regimen comprises intravenous administration of oxaliplatin 85 mg/m ² together with folinic acid 400 mg/m ² followed by fluorouracil 2400 mg/m ² .

In a specific pattern, the FOLFOX dosing regimen is as follows: Day 1: Oxaliplatin 85 mg/m ² and folinic acid 200 mg/m ² are administered as a 2-hour infusion, followed by a bolus of fluorouracil 400 mg/m ² , followed by a 22-hour infusion of fluorouracil 600 mg/m ² ; Day 2: Folinic acid 200 mg/m ² is administered as a 2-hour infusion, followed by a bolus of fluorouracil 400 mg/m ² , followed by a 22-hour infusion of fluorouracil 600 mg/m ² . In a specific pattern, FOLFOX administration is repeated every 2 weeks for 12 cycles.

In certain embodiments, the FOLFOX dosing regimen comprises administering on day 1 of a 2-week cycle: oxaliplatin 85 mg/m ² (e.g., IV infusion over 2 hours) followed by leucovorin 200 mg/m ² (e.g., IV infusion over 2 hours) or simultaneously, and 5-FU 400 mg/m ² (e.g., bolus IV) followed by 5-FU 600 mg/m ² (e.g., continuous infusion over 22 hours). In certain embodiments, the FOLFOX dosing regimen further comprises administering leucovorin 200 mg/m ² (e.g., as an IV infusion over 2 hours) on day 2 of a 2-week cycle, followed by 5-FU 400 mg/m ² (e.g., as an IV bolus), followed by 5-FU 600 mg/m ² (e.g., as a continuous infusion over 22 hours).

In a specific aspect, the FOLFOX dosing regimen comprises oxaliplatin 85 mg/m ² , fluorouracil 400 mg/m ² bolus, folinic acid 400 mg/m ² infused over 2 hours on day 1, followed by fluorouracil 2400 mg/m ² continuous infusion over 46 hours.

If a subject experiences an oxaliplatin-related adverse reaction (also known as an adverse event or AE) after administering one of the relevant methods described above, the method may include adjusting the dosage regimen based on the approved prescribing information for oxaliplatin. Dose adjustments for adverse reactions in advanced colorectal cancer are presented in Table 1.

Management of adverse events may be adjusted based on their severity. Specific dose adjustments and adjuvant measures specified in the prescribing information for oxaliplatin are provided for the following: hypersensitivity reactions, peripheral sensory neuropathy, acute neuropathy, delayed neuropathy, severe myelosuppression, posterior reversible encephalopathy syndrome (PRES), pulmonary toxicity, hepatotoxicity, QT interval prolongation and ventricular arrhythmias, rhabdomyolysis, and hemorrhage.

In certain instances, recommended dose adjustments for oxaliplatin drug-related adverse events follow the FDA-approved prescribing information for eloxatin (Oxaliplatin Injection for Intravenous Use, Revision 6/2023 or latest available at the time of dosing) in Table 1.

In certain instances, if a subject experiences any of the toxicities mentioned in the table above, dose adjustments at the start of subsequent cycles of therapy are made according to Tables 2 and 3 based on the starting dose used in the previous cycle.

In certain instances, if the subject experiences acute and delayed neuropathy, the dose of oxaliplatin is reduced or permanently discontinued according to the severity of the side effect according to the schedule in Table 1 for persistent neurosensory reactions.

In certain embodiments, if the subject experiences persistent Grade 2 neuropathy after receiving oxaliplatin as part of a FOLFOX regimen, the oxaliplatin dose is 65 mg/m ² . In certain embodiments, if the subject experiences persistent Grade 3 neuropathy, oxaliplatin is discontinued. In certain embodiments, if the subject experiences Grade 4 neuropathy, oxaliplatin is discontinued.

In certain aspects, if the subject experiences Grade 4 neutropenia or febrile neutropenia, dosing is delayed until the neutrophil count is ≥1.5× ¹⁰⁹ /L and the platelets are ≥75× ¹⁰⁹ /L. In certain aspects, the oxaliplatin dose is 65 mg/ ^m2 (and is reduced to these values). In certain aspects, if the subject experiences Grade 4 thrombocytopenia, dosing is delayed until the neutrophil count is ≥1.5× ¹⁰⁹ /L and the platelets are ≥75× ¹⁰⁹ /L. In certain aspects, the oxaliplatin dose is 65 mg/ ^m2 (and is reduced to these values).

In certain instances, if a subject experiences a Grade 3 or 4 gastrointestinal adverse reaction after receiving oxaliplatin as part of a FOLFOX regimen, after recovery, the oxaliplatin dose is reduced to 65 mg/m ² while reducing the fluorouracil to 300 mg/m ² as an intravenous bolus and then to 500 mg/m ² as a continuous infusion over 22 hours.

FOLFIRI

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering irinotecan, folinic acid, and fluorouracil. In certain embodiments, the method of treating cancer in a subject further comprises administering to the subject an effective amount of irinotecan, folinic acid (leucovorin), and fluorouracil.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering FOLFIRI. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab and FOLFIRI.

In certain instances, the combination of irinotecan, folinic acid, and fluorouracil is called FOLFOX therapy. FOLFIRI is known by various synonyms, the most common being the FOLFIRI protocol, the FOLFIRI regimen, 5-fluorouracil/folinic acid/irinotecan, and the irinotecan, leucovorin, and fluorouracil combination.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is used in a treatment further comprising administering fluorouracil, leucovorin, and irinotecan (FOLFIRI). In certain embodiments, the subject being treated has mCRC and is wild type for RAS and/or RAF (including Kirsten rat sarcoma (KRAS) and/or B-rapidly accelerated fibrosarcoma (BRAF)). In certain embodiments, the genome of the mCRC is wild type for RAS and/or RAF, including KRAS and/or BRAF. In certain embodiments, the subject being administered FOLFIRI has received only one prior anticancer treatment. In certain embodiments, the prior treatment comprises fluoropyrimidine-oxaliplatin-based chemotherapy with or without bevacizumab.

In certain aspects, the present disclosure provides petosemtamab for use in treating cancer in a subject, wherein the treatment further comprises administering irinotecan, folinic acid, and fluorouracil. In certain aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of petosemtamab, irinotecan, folinic acid, and fluorouracil.

In certain embodiments, the FOLFIRI dosing regimen comprises intravenous administration of irinotecan together with folinic acid followed by administration of fluorouracil.

In certain embodiments, the FOLFIRI dosing regimen comprises intravenous administration of irinotecan 180 mg/m ² together with folinic acid 200 to 400 mg/m ² followed by intravenous administration of fluorouracil 400 to 2400 mg/m ² .

In certain embodiments, the FOLFIRI dosing regimen comprises intravenous administration of irinotecan 180 mg/m ² together with folinic acid 200 mg/m ² or 400 mg/m ² , followed by a bolus of fluorouracil 400 mg/m ² , followed by a continuous infusion of fluorouracil 2400 mg/m ² over 46 hours. In certain embodiments, the FOLFIRI dosing regimen is repeated every 14 days.

In a specific pattern, the FOLFIRI regimen comprises irinotecan 180 mg/m ² by IV infusion over 90 minutes on day 1 of a 2-week cycle, followed by folinic acid (leucovorin) 200 mg/m ² by IV infusion over 2 hours, followed by an IV bolus of fluorouracil 400 mg/m ² , followed by a continuous infusion of fluorouracil 600 mg/m ² over 22 hours. Day 2 of a 2-week cycle - folinic acid (leucovorin) 200 mg/m ² by IV infusion over 2 hours, followed by an IV bolus of fluorouracil 400 mg/m ² , followed by a continuous infusion of fluorouracil 600 mg/m ² over 22 hours.

If a subject experiences a drug-related adverse event (AE) after administering any of the methods described above, the method may include adjusting the dosage regimen based on the approved prescribing information for irinotecan (also known as irinotecan hydrochloride or Camptosar). Dose adjustments for adverse events in advanced colorectal cancer are presented in Table 2.

In certain aspects, the grades and definitions of adverse events (or toxicities or adverse reactions as used herein) follow Table 2, National Cancer Institute (NCI)-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.4.03/v5.0, NCI-CTC v1.0, or the most current information at the time of administration.

In certain instances, recommended dose adjustments for irinotecan drug-related adverse events (AEs) are as described in the National Cancer Institute (NCI) Common Toxicity Criteria (version 1.0).

In certain instances, recommended dose adjustments for irinotecan drug-related adverse events follow the approved prescribing information for Camptosa (Intravenous Injection, Revised 1/2022 or latest information at the time of administration).

In certain instances, if a subject experiences any of the toxicities mentioned in the table above, dose adjustments at the start of subsequent cycles of therapy are made according to Tables 2 and 3 based on the starting dose used in the previous cycle.

In certain instances, the administered dose level is maintained throughout the treatment cycle unless the subject experiences any toxicity. In certain instances, the dose level is maintained at the starting dose throughout subsequent treatment cycles unless the subject experiences any toxicity.

In certain embodiments, if the subject experiences Grade 1 neutropenia (1500 to 1999/mm ³ ), the administered dose level is maintained throughout the therapy cycle. In certain embodiments, the dose level is maintained at the start of subsequent therapy cycles.

In certain embodiments, if the subject experiences Grade 2 neutropenia (1000 to 1499/mm ³ ), the administered dose level is reduced by one dose level during the therapy cycle. In certain embodiments, the dose level is maintained at the start of the subsequent therapy cycle.

In certain embodiments, if a subject experiences Grade 3 neutropenia (500 to 999/mm ³ ), the dose level administered until resolution to Grade 2 or lower is omitted, and the dose is reduced by one dose level during the next cycle of therapy. In certain embodiments, the dose level is reduced by one dose level at the start of the subsequent cycle of therapy.

In certain embodiments, if a subject experiences Grade 4 neutropenia (<500 mm ³ ), the dose level administered until resolution to Grade 2 or lower is omitted, and the dose is reduced by 2 dose levels during the next cycle of therapy. In certain embodiments, the dose level is reduced by 2 dose levels at the start of the subsequent cycle of therapy.

In certain instances, if the subject experiences neutropenic fever, the dose is omitted until it resolves to Grade 2 or lower, and then reduced by 2 dose levels.

In certain instances, if a subject experiences other hematologic toxicities, dose adjustments during and at the start of subsequent cycles are made according to NCI toxicity criteria and are consistent with the recommendations for neutropenia above. In certain instances, if a subject develops leukopenia or thrombocytopenia, dose adjustments during and at the start of subsequent cycles are consistent with the recommendations for neutropenia above.

In certain instances, if a subject experiences Grade 1 diarrhea (2 to 3 stools/day > pretreatment), the dose is delayed until recovery to baseline, and then the same dose is administered during the next treatment cycle. In certain instances, the dose level is maintained at the start of the subsequent treatment cycle.

In certain instances, if a subject experiences Grade 2 diarrhea (4 to 6 stools/day > pretreatment), the dose administered is omitted until recovery to baseline, and then reduced by 1 dose level during the next treatment cycle. In certain instances, the dose level administered is maintained at the start of the subsequent treatment cycle.

In certain embodiments, if a subject experiences Grade 3 diarrhea (7 to 9 stools/day > pretreatment), the dose administered is omitted until recovery to baseline, and then reduced by 1 dose level during the next treatment cycle. In certain embodiments, the dose administered is reduced by 1 dose level at the start of the next treatment cycle.

In certain instances, if a subject experiences Grade 4 diarrhea (10 stools/day > pretreatment), the dose administered is omitted until recovery to baseline, and then reduced by 2 dose levels during the next treatment cycle. In certain instances, the dose administered is reduced by 2 dose levels at the start of the next treatment cycle.

In certain instances, subjects may experience other non-hematologic toxicities. In certain instances, other non-hematologic toxicities do not include alopecia, anorexia, or asthenia.

In certain instances, if the subject experiences other non-hematologic toxicities (Grade 1), the administered dose is maintained throughout the treatment cycle. In certain instances, the administered dose level is maintained at the start of subsequent treatment cycles.

In certain instances, if a subject experiences other non-hematologic toxicities (Grade 2), the dose administered is omitted until the toxicity resolves to Grade 1, and then reduced by one dose level during the next cycle of therapy. In certain instances, the dose level administered is maintained at the start of the subsequent cycle of therapy.

In certain embodiments, if the subject experiences other non-hematologic toxicities (Grade 3), the administered dose is omitted until it resolves to Grade 1, and then reduced by 2 dose levels during the next cycle of therapy. In certain embodiments, the administered dose level is reduced by 1 dose level at the start of the next cycle of therapy.

In certain embodiments, if the subject experiences other non-hematologic toxicities (Grade 4), the administered dose is omitted until the toxicity resolves to Grade 2, and then reduced by two dose levels during the next cycle of therapy. In certain embodiments, the administered dose level is reduced by two dose levels at the start of the next cycle of therapy.

In certain instances, if the subject experiences mucositis or stomatitis, the dose administered is reduced only for 5-FU and not for irinotecan during the treatment cycle. In certain instances, the dose administered is reduced only for 5-FU and not for irinotecan at the start of the subsequent treatment cycle.

multispecific/bispecific antibodies

In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof disclosed herein are multispecific antibodies. In certain aspects, the antibodies are bispecific antibodies. The multispecific or bispecific antibody or functional portions, derivatives, and/or analogs thereof comprise, in certain aspects, a first variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and, in certain aspects, a second variable domain that does not bind EGFR. In certain aspects, the antibody or functional portions, derivatives, and/or analogs thereof monovalently bind EGFR. Furthermore, in certain aspects, the multispecific or bispecific antibody or functional portions, derivatives, and/or analogs thereof comprise a second variable domain that binds LGR5. In certain aspects, the antibody or functional portions, derivatives, and/or analogs thereof comprising a variable domain that binds to the extracellular portion of EGFR and optionally comprising a variable domain that binds to the extracellular portion of LGR5 is or comprises petosemtamab.

EGFR

The epidermal growth factor (EGF) receptor (EGFR, ErbB1, or HER1) belongs to a family of four receptor tyrosine kinases (RTKs), designated Her- or cErbB-1, -2, -3, and -4. EGFR is known by various synonyms, the most common being EGFR. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding, and the remaining two in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands to generate diverse intracellular responses. The primary signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This activates Ras via the Grb2-binding Ras-guanine nucleotide exchange factor, Son of Sevenless (SOS). Furthermore, the PI3-kinase-Akt signaling pathway is also activated by EGFR, and this activation is significantly enhanced in the presence of co-expression of ErbB-3 (HER3). EGFR is associated with several human epithelial malignancies, particularly breast, bladder, non-small cell lung, colon, ovarian, head and neck, and brain cancers. Activating mutations in this gene, as well as overexpression of the receptor and its ligand, have been found, resulting in an autocrine activation loop. Therefore, these RTKs have been widely used as targets for cancer therapy. Both small molecule inhibitors targeting RTKs and monoclonal antibodies (mAbs) targeting the extracellular ligand-binding domain have been developed, but have achieved limited clinical success to date, primarily in selected patient populations. The database accession number for the human EGFR protein and the gene encoding it is GenBank NM_005228.3. This accession number is provided primarily to provide additional tools for targeting the EGFR protein; the actual sequence of the EGFR protein bound by antibodies can vary due to mutations in the encoding gene, such as those found in some cancers.

When EGFR is mentioned herein, it refers to human EGFR unless otherwise specified. The variable domain antigen-binding site that binds EGFR binds to EGFR and various variants thereof, such as those expressed in some EGFR-positive tumors.

In certain aspects, the EGFR is human EGFR. The EGFR bound by the antibodies or functional portions, derivatives, and/or analogs thereof of the present invention includes wild-type EGFR as well as EGFR having oncogenic mutations. In certain aspects, the oncogenic mutations are activating EGFR mutations. In certain aspects, such mutations do not conformationally alter the epitope bound by the antibodies of the present disclosure. In certain aspects, the EGFR mutations of the present disclosure include mutations, such as exon 18 mutations, such as G719A, G719C, 2E709_T710D, E709A, G719S; exon 19 deletion mutations, such as deletions of LREA or VAIKEL; exon 19 point mutations, such as G735S, P753L, L747S, D761Y; In-frame exon 20 insertion mutations of 1 to 7 amino acids, exon 20 point mutations, such as V765A, T783A, V774A, S784P, V769M, T790M; exon 21 mutations, such as L858R, T854A, A871E, L861A, L861C, L861S, V843I, or P848L. The antibodies of the present disclosure bind to an epitope that is not adjacent to said mutations. In particular, the EGFR mutation is S492R, which results in loss of cetuximab binding to EGFR. The antibodies of the present disclosure bind to an epitope that is different from the epitope recognized by cetuximab.

Without wishing to be bound by any theory, it is believed that amino acid residues I462; G465; K489; I491; N493; and C499, as depicted in FIG. 2, are involved in epitope binding by the antibodies of the present disclosure. In certain embodiments, binding involvement is determined by observing reduced binding of a variable domain to EGFR having one or more of the amino acid residue substitutions selected from I462A; G465A; K489A; I491A; N493A; and C499A.

In one aspect, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420 to 480 of the sequence depicted in FIG. 2. In a specific aspect, binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions within EGFR: I462A; G465A of EGFR; K489A; I491A; N493A; and C499A. In a specific aspect, binding of the antibody to human EGFR prevents EGF from binding to the receptor. In a specific aspect, the epitope on EGFR is a conformational epitope. In one aspect, the epitope is located within amino acid residues 420 to 480 of the sequence depicted in FIG. 2 or amino acid residues 430 to 480 of the sequence depicted in FIG. 2. In certain embodiments, the epitope is located within amino acid residues 438 to 469 of the sequence depicted in FIG. 2.

Without wishing to be bound by theory, the contact residues of the epitope, i.e., the positions where the variable domain contacts human EGFR, are believed to be I462; K489; I491; and N493. Amino acid residues G465 and C499 are indirectly involved in binding of the antibody to EGFR.

In an exemplary method, CHO cells express EGFR on their cell membranes, or express mutants comprising one or more substitutions selected from I462A; G465A; K489A; I491A; N493A; and C499A. A test antibody is contacted with the CHO cells and binding of the antibody to the cells is compared. The test antibody binds to the epitope if it binds to EGFR, and to a lesser extent if it binds to EGFR having the I462A; G465A; K489A; I491A; N493A; and C499A substitutions. It is preferred to compare binding to a panel of mutants, each comprising a single alanine residue substitution. Such binding studies are well known in the art. Often, the panel comprises single alanine substitution mutants encompassing essentially all amino acid residues. For EGFR, the panel only needs to include the extracellular portion of the protein and, if using cells, the portion required for association with the cell membrane.

Although expression of a specific mutant may be reduced, this is detected by one or more EGFR antibodies that bind to different regions. If expression of this control antibody is also reduced, the membrane protein level or folding of this specific mutant is reduced. Binding characteristics for a panel of test antibodies determine whether the test antibody exhibits reduced binding to mutants with the I462A, G465A, K489A, I491A, N493A, and C499A substitutions.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative, and/or analog thereof comprising a first variable domain that binds to the extracellular portion of EGFR, wherein the antibody is a monovalent antibody that does not comprise a second variable domain, or wherein the antibody comprises an EGFR-binding variable domain as the only variable domain. In certain aspects, the present disclosure provides an antibody or functional portion, derivative, and/or analog thereof comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that does not bind EGFR. In certain aspects, the antibody or functional portion, derivative, and/or analog thereof monovalently binds EGFR. In certain aspects, the antibody or functional portion, derivative, and/or analog thereof. In certain aspects, the antibody comprises a second variable domain that binds LGR5.

In certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a first variable domain that binds to the extracellular portion of EGFR and a second variable domain that binds to the extracellular portion of LGR5.

LGR5

The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G-protein-coupled receptors. Several members of this family are known to be involved in the WNT signaling pathway, notably LGR4, LGR5, and LGR6.

LGR5 is leucine-rich repeat-containing G-protein coupled receptor 5. Alternative names for this gene or protein are leucine-rich repeat-containing G-protein coupled receptor 5, leucine-rich repeat-containing G-protein coupled receptor 5, G-protein coupled receptor HG38, G-protein coupled receptor 49, G-protein coupled receptor 67, GPR67, GPR49, orphan G-protein coupled receptor HG38, G-protein coupled receptor 49, GPR49, HG38, and FEX. A protein or antibody of the present disclosure that binds to LGR5 binds to human LGR5. An LGR5 binding protein or antibody of the present disclosure may, but does not necessarily, bind to human and other mammalian orthologs due to sequence and tertiary structure similarities between these orthologs. The database accession numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12; NT_029419.13; NC_018923.2; NP_001264155.1; NP_001264156.1; NP_003658.1). The accession numbers are provided primarily to provide additional methods for identifying LGR5 as a target, as the actual sequence of the LGR5 protein to be bound may vary due to mutations in the encoding gene, such as those occurring in some cancers. The LGR5 antigen-binding site binds LGR5 and various variants thereof, such as those expressed by some LGR5-positive tumor cells.

An antibody or functional portion, derivative, and/or analog thereof as described herein comprises a variable domain that binds to the extracellular portion of LGR5. In certain aspects, the second variable domain binds LGR5. In certain aspects, the LGR5 is human LGR5. A multispecific or bispecific antibody or functional portion, derivative, and/or analog thereof as described herein comprises a variable domain that binds to the extracellular portion of the human epidermal growth factor (EGF) receptor and, in certain aspects, a variable domain that binds to human LGR5.

In certain aspects, the antibodies or functional portions, derivatives, and/or analogs thereof described herein comprise a variable domain that binds to the extracellular portion of an epidermal growth factor (EGF) receptor and interferes with binding of EGF to the receptor, and a variable domain that binds LGR5, wherein interaction of the antibody with LGR5 on an LGR5-expressing cell does not block binding of Rspondin (RSPO) to LGR5. Methods for determining whether an antibody blocks or does not block binding of Rspondin to LGR5 are described in WO2017/069628, which is incorporated herein by reference.

In a specific aspect, the variable domain that binds to the extracellular portion of LGR5 binds to an epitope located within amino acid residues 21 to 118 of the sequence of Figure 1, of which amino acid residues D43, G44, M46, F67, R90 and F91 are involved in binding of the antibody to the epitope.

In a specific aspect, the LGR5 variable domain is a variable domain in which one or more of the amino acid residue substitutions within LGR5, D43A, G44A, M46A, F67A, R90A and F91A, reduce binding of the variable domain to LGR5.

In certain embodiments, the epitope on LGR5 is a conformational epitope. In certain embodiments, the epitope is located within amino acid residues 40 to 95 of the sequence of Figure 1. In certain embodiments, binding of the antibody to LGR5 is reduced by one or more of the following amino acid substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

Without wishing to be bound by theory, M46, F67, R90, and F91 of LGR5, as depicted in Figure 1, are believed to be contact residues for the antigen-binding site of the variable domain mentioned above, i.e., the variable domain that binds the LGR5 epitope. While the amino acid residue substitutions D43A and G44A may reduce antibody binding because they are also contact residues, it is also possible that these amino acid residue substitutions induce a (slight) conformational change in the portion of LGR5 that has one or more of the other contact residues (i.e., positions 46, 67, 90, or 91), resulting in reduced antibody binding due to the conformational change. Epitopes are characterized by the amino acid substitutions mentioned. Whether an antibody binds to the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 on the cell membrane or express mutants comprising one or more of the following substitutions: M46A, F67A, R90A, or F91A. A test antibody is contacted with the CHO cells, and binding of the antibody to the cells is compared. The test antibody binds to the epitope if it binds to LGR5, and to a lesser extent if it binds to LGR5 with the M46A, F67A, R90A, or F91A substitutions. It is preferred to compare binding to a panel of mutants, each comprising a single alanine residue substitution. Such binding studies are well known in the art. Often, the panel comprises single alanine substitution mutants encompassing essentially all amino acid residues. For LGR5, the panel need only include the extracellular portion of the protein and, if cells are used, the portion required for cell membrane association. Although expression of a particular mutant may be reduced, this is readily detected by one or more LGR5 antibodies that bind to different regions. If expression of this control antibody is also reduced, then the membrane protein level or folding of this particular mutant is reduced. The binding characteristics of the test antibody to the panel indicates whether the test antibody exhibits reduced binding to the mutants having the M46A, F67A, R90A, or F91A substitutions, and thus whether the test antibody is an antibody of the invention. Reduced binding to the mutants having the M46A, F67A, R90A, or F91A substitutions also identifies an epitope located within amino acid residues 21 to 118 of the sequence of Figure 1. In a specific aspect, the panel includes a D43A substitution mutant; and a G44A substitution mutant of both. Antibodies having the VH sequence of the VH of MF5816 exhibit reduced binding to these substitution mutants.

Where accession numbers or alternative names for proteins/genes are provided herein, this is primarily to provide additional methods of identifying the protein as a target, and the actual sequence of the target protein bound by the antibodies of the present disclosure may vary, for example, due to mutations and/or alternative splicing of the encoding gene, such as occurs in some cancers. A target protein is bound by an antibody as long as the epitope is present on the protein and the antibody can access the epitope.

Antibodies that bind to EGFR and LGR5

The present disclosure further provides an antibody having a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the LGR5 variable domain binds to an epitope of LGR5 located within amino acid residues 21 to 118 of the sequence of FIG. 1.

Suitable variable domains that bind to the extracellular portion of EGFR and suitable variable domains that bind to the extracellular portion of LGR5 are disclosed herein. In certain aspects, the first variable domain comprises at least the CDR3 sequence, or at least CDR1, CDR2, and CDR3 sequences, of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370; MF3755; MF4280, or MF4289, as depicted in FIG. 3 . In certain aspects, the second variable domain comprises at least the CDR3 sequence, or at least CDR1, CDR2, and CDR3 sequences, of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, as depicted in FIG. 3 .

In certain aspects, the variable domain that binds human EGFR is a variable domain having a heavy chain variable region comprising a CDR3 sequence of the VH of MF3755 as depicted in FIG. 3 or a CDR3 sequence that differs from the CDR3 sequence of the VH of MF3755 as depicted in FIG. 3 by at most 3, or at most 2, or not more than 1 amino acid.

In certain embodiments, the variable domain that binds human EGFR is a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as depicted in FIG. 3; or the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as depicted in FIG. 3 with at most three, at most two or at most one amino acid substitution.

In certain embodiments, the variable domain that binds human EGFR is a variable domain having a heavy chain variable region comprising the amino acid sequence of the VH chain of MF3755 as depicted in FIG. 3; or having up to 15 (or in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments, 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof relative to the VH chain of MF3755.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5, wherein the heavy chain variable region of the variable domain comprises a CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3, or wherein the heavy chain variable region of the variable domain comprises a heavy chain CDR3 sequence that differs by at most 3, or at most 2, or at most 1 amino acid from the CDR3 sequence of a VH selected from the group consisting of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. In certain aspects, the variable domain comprises at least MF3370; MF3755; It comprises a heavy chain variable region comprising the CDR3 sequence of MF4280 or MF4289.

In certain aspects, the variable domain comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of at least MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3, or a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences that differ by at most 3, or at most 2, or at most 1 amino acid from the CDR1, CDR2 and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of at least MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. In certain aspects, the variable domain comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of at least MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3. In certain embodiments, the heavy chain variable region is MF3755. In certain embodiments, the heavy chain variable region is MF4280.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5, wherein the variable domain that binds the extracellular portion of EGFR is a heavy chain variable region comprising the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3; or the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3 having up to fifteen 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain of MF3370; MF3755; MF4280 or MF4289.

In certain embodiments, the antibody comprises a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5, wherein the EGFR binding variable domain has CDR3, CDR1, CDR2 and CDR3 and/or VH sequences as set forth above, or at least a CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3 or MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; as depicted in FIG. 3. Or a variable domain that binds LGR5 comprising a heavy chain CDR3 sequence that differs by at most 3, or at most 2, or at most 1 amino acid from the CDR3 sequence of a VH selected from the group consisting of MF5790; MF5803; MF 5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818. In certain aspects, the variable domain comprises a heavy chain variable region comprising the CDR3 sequence of at least MF5790; MF5803; MF 5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3 .

In certain embodiments, the LGR5 binding variable domain comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of at least MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3 or at least MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; as depicted in FIG. 3. Or comprises a heavy chain CDR1, CDR2 and CDR3 sequence that differs by at most 3, or at most 2, or at most 1 amino acid from the CDR1, CDR2 and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5818. In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3. In certain aspects, the heavy chain variable region is MF5790; MF5803; MF5814; MF5816; MF5817; or MF5818. In certain aspects, the heavy chain variable region is MF5790; MF5814; MF5816; or MF5818. In certain embodiments, the heavy chain variable region is MF5814, MF5818, or MF5816. In certain embodiments, the heavy chain variable region is MF5816. In certain embodiments, the heavy chain variable region is MF5818.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5, wherein the variable domain that binds the extracellular portion of LGR5 has an amino acid sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, as depicted in FIG. 3; or MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; Or a heavy chain variable region comprising the amino acid sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, as shown in FIG. 3, having up to 15 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or combinations thereof for the VH chain of MF5818.

Antibodies comprising the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof have been shown to be more effective when used to inhibit the growth of cancers or cells that respond to EGFR ligands. In the context of bispecific or multispecific antibodies, an antibody arm comprising the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof combines well with an antibody arm comprising the heavy chain variable region MF5818 or one or more variable domains having one or more CDRs thereof.

Antibodies comprising the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof have been shown to be more effective when used to inhibit the growth of cancers or cells that respond to EGFR ligands. In the context of bispecific or multispecific antibodies, an antibody arm comprising the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof combines well with an antibody arm comprising the heavy chain variable region MF5816 or one or more variable domains having one or more CDRs thereof.

The VH chain of the variable domain that binds EGFR or LGR5 can have one or more amino acid substitutions relative to the sequence depicted in FIG. 3. In certain aspects, the VH chain has the amino acid sequence of the EGFR or LGR5 VH of FIG. 3, which has at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain aspects 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof relative to the VH chain sequence of FIG.

A CDR sequence may have one or more amino acid residue substitutions relative to the CDR sequences in the drawing. These one or more substitutions are performed for optimization purposes, such as improving the binding strength or stability of the antibody. Optimization is performed, for example, by mutagenesis procedures, wherein the stability and/or binding affinity of the resulting antibody is tested, for example, and an improved EGFR-specific CDR sequence or LGR5-specific CDR sequence is selected. One skilled in the art is well capable of generating antibody variants comprising at least one altered CDR sequence according to the present disclosure. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include substituting one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and substituting one polar residue for another polar residue, such as substituting arginine for lysine, glutamic acid for aspartic acid, or glutamine for aspartic acid.

In certain aspects, the amino acid substitutions of up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain aspects 1, 2, 3, 4, or 5) amino acids in a VH or VL as described herein are conservative amino acid substitutions. In certain aspects, the amino acid insertions, deletions, and substitutions in a VH or VL as described herein are not in the CDR3 region. In certain aspects, the amino acid insertions, deletions, and substitutions are also not in the CDR1 and CDR2 regions. In certain aspects, the amino acid insertions, deletions, and substitutions are also not in the FR4 region.

In certain aspects, the insertion, deletion, substitution or combination thereof is not present in the CDR3 region of the VH chain, in certain aspects it is not present in the CDR1, CDR2 or CDR3 region of the VH chain, and in certain aspects it is not present in the FR4 region.

In certain embodiments, the insertion, deletion, substitution or combination thereof is not present in the CDR1, CDR2 and CDR3 regions of the VH chain.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5790 as shown in Figure 3; or

- An amino acid sequence of VH chain MF5790 as depicted in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5803 as shown in Figure 3; or

- An amino acid sequence of VH chain MF5803 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 4, 7, 8, 9 or 10) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5814 as shown in Figure 3; or

- An amino acid sequence of VH chain MF5814 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5816 as shown in Figure 3; or

- An amino acid sequence of VH chain MF5816 as depicted in Figure 3, having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof compared to the above VH.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5817 as shown in Figure 3; or

- Amino acid sequence of VH chain MF5817 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof compared to the above VH.

In certain aspects, the present disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising:

- Amino acid sequence of VH chain MF3755 as shown in Figure 3; or

- an amino acid sequence of VH chain MF3755 as shown in Figure 3 having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH; and

Here, the VH chain of the variable domain that binds LGR5 comprises:

- Amino acid sequence of VH chain MF5818 as shown in Figure 3; or

- An amino acid sequence of VH chain MF5818 as shown in Figure 3, having up to 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof compared to the above VH.

In certain embodiments, the antibody is petosemtamab (see WHO Drug Information, Recommended INN: List 83. Vol. 34, No. 1, 2020, Pg75-77). In certain embodiments, the functional portion, derivative, and/or analog thereof is a functional portion, derivative, and/or analog of petosemtamab. In certain embodiments, the EGFR/LGR5 antibody of the present disclosure is or comprises petosemtamab.

In a specific aspect, the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of a variable region selected from the group consisting of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3 , wherein the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of a variable region selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3 .

In a specific aspect, the variable domain that binds the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1, CDR2 and CDR3 sequences of the variable region of MF3755 as depicted in FIG. 3 , while the variable domain that binds the extracellular portion of LGR5 comprises a heavy chain variable region comprising the CDR1, CDR2 and CDR3 sequences of the variable region of MF5816 as depicted in FIG. 3 .

In a specific aspect, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3; or the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5790; MF5803; MF5805; MF5808; MF5809 as depicted in FIG. 3. MF5814; MF5816; MF5817; or MF5818; or a VH chain MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof.

In a specific aspect, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in FIG. 3; or the amino acid sequence of VH chain MF3755 as depicted in FIG. 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof, relative to said VH; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in FIG. 3; Or comprises the amino acid sequence of VH chain MF5816 as shown in Figure 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof, for said VH.

In a specific aspect, the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3; and the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3.

In a specific aspect, the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in FIG. 3; and the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in FIG. 3.

In certain embodiments, both the variable domain that binds EGFR and the variable domain that binds LGR5 comprise CDR1, CDR2 and CDR3 regions of the light chain variable region as depicted in FIG. 4B .

In a specific aspect, both the variable domain that binds EGFR and the variable domain that binds LGR5 comprise a light chain variable region as depicted in FIG. 4B , wherein the variable light chain region comprises 0 to 10 amino acid insertions, deletions, substitutions, additions or combinations thereof, provided that the amino acid insertions, deletions and substitutions are not present in the CDR1, CDR2 and CDR3 light chain variable regions.

Additional variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries containing rearranged human IGKV1-39/IGKJ1 VL regions (De Kruif et al. Biotechnol Bioeng. 2010 (106)741-50) and collections of VH regions that incorporate amino acid substitutions into the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein as previously described (e.g., WO2017/069628). Phage encoding Fab regions that bind EGFR or LGR5 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions or additions that retain antigen binding.

In certain embodiments, the light chain variable regions of the VH/VL EGFR and LGR5 variable domains of the EGFR/LGR5 antibody can be the same or different. In certain embodiments, the VL region of the VH/VL EGFR variable domain of the EGFR/LGR5 antibody is similar to the VL region of the VH/VL LGR5 variable domain. In certain embodiments, the VL regions in the first and second VH/VL variable domains are identical.

In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of the EGFR/LGR5 antibody comprises a common light chain variable region. In some aspects, the common light chain variable region of one or both of the VH/VL variable domains comprises a germline IgVκ1-39 variable region V-segment. In certain aspects, the light chain variable region of one or both of the VH/VL variable domains comprises a kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39. The external ID of the gene is HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. Amino acid sequences for suitable V-regions are provided in FIG. 4 . The V-regions can be combined with one of five J-regions. In certain embodiments, the J-regions are jk1 and jk5, and the combined sequences are designated IGKV1-39/jk1 and IGKV1-39/jk5; alternative designations are IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01 (nomenclature according to the IMGT database World Wide Web imgt.org). In certain embodiments, the light chain variable region of one or both of the VH/VL variable domains comprises a kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1- 39*01/IGJκ1*05 (as described in FIG. 4 ).

In certain embodiments, the light chain variable region of FIG. 4d) comprises LCDR1, LCDR2, and LCDR sequences. These sequences can be determined or annotated by a skilled artisan using an annotation system such as, for example, IMGT, Chothia, Kabat, or other suitable annotation system.

The CDRs and framework regions of antibody heavy and light chains are described, for example, by Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991); Kabat et al., J. Biol. Chem. 252:6609-6616 (1977)), IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25:206-21 1 1997), Chothia (Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; Al-Lazikani et al., J. Mol. Biol. 273:927-948, The nomenclature of CDRs has been described and defined in the art using a number of different systems, including the nomenclature of Honnegher and Pluckthun (Honnegher and Pluckthun, J. Mol. Biol. 309: 657-670, 2001), MacCallum (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)), and Lefranc (Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003)). In general, antibodies exhibit their properties regardless of which numbering system is used, so which numbering system is used is not important. Given the amino acid sequence of a variable region, one of ordinary skill in the art can readily determine the CDRs of that variable region based on one of the various numbering systems presented above. Accordingly, the present disclosure includes defining CDRs according to each numbering system available to those skilled in the art. In particular, the present disclosure includes defining CDRs according to the numbering systems of Kabat, IMGT, and Chothia. In certain aspects, the heavy chain CDRs are as defined using Kabat, and the light chain CDRs are as defined using IMGT. The amino acids in the constant region are indicated according to the EU numbering system.

In certain embodiments, the light chain variable region comprises or is one or both VH/VL variable domains present in an EGFR-binding antibody of the present disclosure, and comprises LCDR1, LCDR2, and LCDR sequences. Such sequences can be determined or annotated by one of ordinary skill in the art using an annotation system such as, for example, IMGT, Chothia, Kabat, or other suitable annotation system.

In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of the bispecific antibody of the present disclosure comprises LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4 ), LCDR2 comprising the amino acid sequence AAS (as depicted in FIG. 4 ), and LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4 ) (i.e., CDRs of IGKV1-39 according to IMGT). In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of the EGFR/LGR5 binding antibody of the present disclosure comprises LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4 ), LCDR2 comprising the amino acid sequence AAS (as depicted in FIG. 4 ), and LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4 ).

In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of the EGFR/LGR5 bispecific antibody of the present disclosure comprises LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4 ), LCDR2 comprising the amino acid sequence AAS (as depicted in FIG. 4 ), and LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4 ) (i.e., CDRs of IGKV1-39 according to IMGT). In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of the EGFR/LGR5 antibody comprises LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4 ), LCDR2 comprising the amino acid sequence AAS (as depicted in FIG. 4 ), and LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4 ).

In certain aspects, one or both of the VH/VL variable domains of the EGFR/LGR5 antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90% identical, in certain aspects at least 95% identical, in certain aspects at least 97% identical, in certain aspects at least 98% identical, in certain aspects at least 99% identical, or in certain aspects 100% identical, to the amino acid sequence set forth in FIG. 4 .

For example, the variable light chain of one or both of the VH/VL variable domains of an EGFR/LGR5 antibody can have 0 to 10, and in certain aspects 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof, relative to the sequence of FIG. 4. In certain aspects, the light chain variable region of one or both of the VH/VL variable domains of an EGFR/LGR5 antibody comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, in certain aspects 0 to 3, in certain aspects 0 to 2, in certain aspects 0 to 1, and in certain aspects 0 amino acid insertions, deletions, substitutions, additions relative to the amino acid sequences provided, or combinations thereof.

Additionally, the light chain variable region of one or both of the VH/VL variable domains of the EGFR/LGR5 antibody comprises an amino acid sequence as shown in FIG. 4 . In certain aspects, both VH/VL variable domains of the EGFR/LGR5 antibody comprise the same VL region. In certain aspects, the VL of both VH/VL variable domains of the EGFR/LGR5 bispecific antibody comprises the amino acid sequence shown in FIG. 4 . In certain aspects, the VL of both VH/VL variable domains of the EGFR/LGR5 bispecific antibody comprises the amino acid sequence shown in FIG. 4 .

In certain aspects, the antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR is a bispecific antibody having two variable domains, one of which binds EGFR as described herein and the other of which binds LGR5. The antibody or functional portion, derivative and/or analog thereof can be provided in a number of formats. Several different formats of bispecific antibodies are known in the art and are reviewed by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47; MAbs, 2012 Mar-Apr;4(2):182-97) and Spiess et al. (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015) http: //dx.doi.org/10.1016/j.molimm.2015.01.003), each of which is incorporated herein by reference. For example, a bispecific antibody format, rather than a classical antibody having two VH/VL combinations, has a variable domain comprising at least a heavy chain variable region and a light chain variable region. This variable domain can be linked to a single-chain Fv fragment, a monobody, a VH, and a Fab fragment that provides a second binding activity.

In certain aspects, the antibody or functional portion, derivative and/or analog thereof is of a human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain aspects, the antibody is of a human IgG1 subclass. Full-length IgG antibodies are preferred because of their favorable half-life and low immunogenicity. Thus, in certain aspects, the bispecific antibody is a full-length IgG molecule. In certain aspects, the bispecific antibody is a full-length IgG1 molecule. Thus, in certain aspects, the bispecific antibody comprises a crystallizable fragment (Fc). In certain aspects, the Fc of the EGFR/LGR5 bispecific antibody comprises a human constant region. The Fc or constant region of the EGFR/LGR5 bispecific antibody may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. For example, each Fab-arm of a bispecific antibody may additionally comprise an Fc-region comprising modifications that promote bispecific antibody formation, promote stability, and/or promote other properties described herein.

EGFR signaling inhibition

The antibody or functional part, derivative, and/or analog thereof as described herein preferably interferes with the binding of a ligand for EGFR to EGFR. The term "interfering with binding" as used herein means that binding of the antibody or functional part, derivative, and/or analog thereof to EGFR competes with the ligand for binding to the EGF receptor. The antibody or functional part, derivative, and/or analog thereof may reduce ligand binding, displace the ligand if the ligand is already bound to the EGF receptor, or at least partially prevent the ligand from binding to the EGF receptor, for example, through steric hindrance.

In certain aspects, the EGFR antibodies disclosed herein inhibit EGFR ligand-induced signaling, as measured by ligand-induced growth of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058) or ligand-induced cell death of A431 cells (ATCC CRL-1555), respectively. EGFR binds to a number of ligands and can stimulate the growth of the BxPC3 cells or BxPC3-luc2 cells mentioned. In the presence of an EGFR ligand, the growth of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced growth of BxPC3 cells can be measured by comparing the growth of cells in the absence and presence of the ligand. In certain aspects, a preferred EGFR ligand for measuring EGFR ligand-induced growth of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced growth is preferably measured using a saturating amount of the ligand. In certain embodiments, EGF is used in an amount of 100 ng/ml of culture medium. In certain embodiments, the EGF is EGF from R&D systems, catalog numbers 396-HB and 236-EG (see also WO2017/069628, which is incorporated herein by reference).

In certain embodiments, the EGFR antibody disclosed herein preferably inhibits EGFR ligand-induced growth of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR binds to a number of ligands and can stimulate the growth of the BxPC3 cells or BxPC3-luc2 cells mentioned above. In the presence of the ligand, the growth of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced growth of BxPC3 cells can be measured by comparing the growth of cells in the absence and presence of the ligand. In certain embodiments, a preferred EGFR ligand for measuring EGFR ligand-induced growth of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced growth is preferably measured using a saturating amount of the ligand. In certain embodiments, EGF is used at an amount of 100 ng/ml of culture medium. In certain embodiments, the EGF is EGF from R&D systems, catalog numbers 396-HB and 236-EG (see also WO2017/069628, which is incorporated herein by reference).

To avoid any doubt, references to cell growth herein refer to changes in cell number. Growth inhibition refers to a decrease in the number of cells that would otherwise be obtained. Growth enhancement refers to an increase in the number of cells that would otherwise be obtained. Cell growth typically refers to cell proliferation.

Whether an antibody described herein inhibits signal transduction or growth in a multispecific fashion is preferably determined by the methods described herein using a monospecific monovalent or monospecific bivalent version of the antibody. In certain embodiments, the antibody has a binding site for a receptor for which signal transduction is to be determined. A monospecific monovalent antibody may have variable domains with unrelated binding specificities, such as tetanus toxoid specificity. In certain embodiments, the antibody is a bivalent monospecific antibody in which the antigen-binding variable domain consists of a variable domain that binds to a member of the EGF-receptor family.

Nucleic acids and cells

Bispecific antibodies are typically produced by cells expressing nucleic acid(s) encoding the antibody. Thus, in some aspects, the bispecific EGFR/LGR5 antibodies disclosed herein are produced by providing a cell comprising one or more nucleic acids encoding the heavy and light chain variable regions and constant regions of the bispecific EGFR/LGR5 antibody. The cell is preferably an animal cell, such as a mammalian cell, a primate cell, or, in certain aspects, a human cell. Suitable cells are any cells capable of comprising, and preferably producing, an EGFR/LGR5 bispecific antibody.

Suitable cells for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. Various institutions and companies have developed cell lines for large-scale antibody production, for example, for clinical use. Non-limiting examples of such cell lines include CHO cells, NS0 cells, or PER-C6 cells. In certain embodiments, the cells are human cells. In certain embodiments, the cells are transformed with the adenovirus E1 region or a functional equivalent thereof. A preferred example of such a cell line is the PER-C6 cell line or an equivalent thereof. In certain embodiments, the cells are CHO cells or variants thereof. In certain embodiments, the variants utilize a glutamine synthetase (GS) vector system for antibody expression. In certain embodiments, the cells are CHO cells.

In some embodiments, the cell expresses different light and heavy chains that constitute an EGFR/LGR5 bispecific antibody. In certain embodiments, the cell expresses two different heavy chains and at least one light chain. In certain embodiments, the cell expresses a "common light chain" as described herein to reduce the number of different antibody species (different combinations of heavy and light chains). For example, each VH region can be cloned into an expression vector using methods known in the art for the production of bispecific IgGs (WO2013/157954; incorporated herein by reference) together with a rearranged human IGKV1 39/IGKJ1 (huVκ1 39) light chain, which has previously been shown to be capable of pairing with more than one heavy chain, thereby generating antibodies with different specificities, facilitating the production of bispecific molecules (De Kruif et al. J. Mol. Biol. 2009 (387) 548 58; WO2009/157771).

Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies (i.e., antibodies with identical heavy and light chain combinations). Several methods have been disclosed to favor the production of bispecific antibodies over the production of individual monospecific antibodies. This is typically achieved by modifying the constant regions of the heavy chains to favor heterodimerization (i.e., dimerization with heavy chains of different heavy/light chain combinations) over homodimerization. In certain aspects, the bispecific antibodies of the present disclosure comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains are described in the art. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. The art describes various ways in which such heterodimerization of heavy chains can be achieved.

One preferred method for producing EGFR/LGR5 bispecific antibodies is disclosed in US 9,248,181 and US 9,358,286. Specifically, preferred mutations for producing essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain (the 'KK-variant' heavy chain) and the amino acid substitutions L351D and L368E in the second CH3 domain (the 'DE-variant' heavy chain), or vice versa. As previously described, the DE-variant and the KK-variant preferentially pair to form heterodimers (so-called 'DEKK' bispecific molecules). Homodimerization of DE-variant heavy chains (DEDE homodimers) or KK-variant heavy chains (KKKK homodimers) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

Thus, in a particular aspect, a heavy chain/light chain combination comprising a variable domain that binds EGFR comprises a DE variant of the heavy chain. In a particular aspect, a heavy chain/light chain combination comprising a variable domain that binds LGR5 comprises a KK variant of the heavy chain.

Candidate EGFR/LGR5 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be assessed by flow cytometry (according to the FACS procedure previously described in WO2017/069628). In certain embodiments, binding of the candidate EGFR/LGR5 bispecific antibody to LGR5 on CHO cells is demonstrated by flow cytometry performed according to standard procedures known in the art. Binding to CHO cells is compared to CHO cells not transfected with expression cassettes for EGFR and/or LGR5. Binding of the candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct; LGR5 monospecific antibodies and EGFR monospecific antibodies, as well as an unrelated IgG1 isotype control mAb, are included in the assay as controls (e.g., antibodies that bind LGR5 and another antigen such as tetanus toxoid (TT).

The affinity of the candidate EGFR/LGR5 bispecific antibody for its LGR5 target and the EGFR Fab for its target can be measured by surface plasmon resonance (SPR) technology using a BIAcore T100. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog no. 555784) is conjugated to the surface of a CM5 sensor chip using free amine chemistry (NHS/EDC). The bispecific antibody is then captured on the sensor surface. Subsequently, recombinant purified antigen human EGFR (Sino Biological Inc., catalog no. 11896-H07H) and human LGR5 proteins are spread over a range of concentrations to measure the on-rate and off-rate. After each cycle, the sensor surface is regenerated by an HCl pulse, and the bispecific antibody is recaptured. From the obtained sensorgrams, on-rate and off-rate and affinity values for binding to human LGR5 and EGFR were determined using BIAevaluation software as previously described for CD3 in US 2016/0368988.

ADCC

Antibodies disclosed herein are typically, in certain embodiments, bispecific full-length antibodies of the human IgG subclass. In certain embodiments, the antibodies are of the human IgG1 subclass. Such antibodies have superior ADCC properties, which can be enhanced, if desired, by techniques known in the art, have favorable half-lives when administered in vivo to humans, and CH3 engineering techniques exist that can provide modified heavy chains that preferentially form heterodimers over homodimers when co-expressed in clonal cells.

If the antibody itself has low ADCC activity, its ADCC activity can be improved by modifying its constant region. Another way to improve ADCC activity is to enzymatically interfere with the glycosylation pathway to reduce fucose. Several in vitro methods exist to determine the efficacy of antibodies or effector cells in inducing ADCC. These include the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a specific surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with the antibody-labeled target cells. Target cell lysis is subsequently measured by the release of intracellular label using a scintillation counter or spectrophotometrically.

Bispecific antibodies as described herein preferably have enhanced ADCC. In certain aspects, the bispecific antibodies may be afucosylated. In certain aspects, the bispecific antibodies preferably comprise a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region compared to the same antibody produced in normal CHO cells. Lower fucose levels are associated with increased CD16 (FcγRIIIa) binding on NK effector cells, thereby increasing ADCC activity. In certain aspects, the bispecific antibodies of the present disclosure can eliminate tumor cells through opsonization and subsequent natural killer (NK) cell-mediated ADCC activity and complement-dependent cytotoxicity (CDC) activity, in addition to direct antitumor activity.

An antibody comprising a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 may further comprise one or more additional variable domains capable of binding one or more additional targets. In certain aspects, the additional target is a protein, such as a membrane protein, comprising an extracellular portion. As used herein, a membrane protein is a cell membrane protein, for example, a protein present in the outer membrane of a cell, i.e., the membrane that separates a cell from the outside world. A membrane protein has an extracellular portion. A membrane protein is present at least on a cell if it comprises a transmembrane region present in the cell membrane of the cell.

Antibodies with more than two variable domains are known in the art. For example, it is possible to attach additional variable domains. In certain embodiments, antibodies with three or more variable domains are multivalent multimeric antibodies, as described in PCT/NL2019/050199, which is incorporated herein by reference.

In certain aspects, the antibody is a bispecific antibody comprising two variable domains, wherein one variable domain binds to the extracellular portion of EGFR and the other variable domain binds to the extracellular portion of LGR5. In certain aspects, the variable domains are preferably variable domains as described herein.

The functional portion of an antibody described herein comprises at least a variable domain that binds the extracellular portion of EGFR and a variable domain that binds the extracellular portion of LGR5 as described herein. Accordingly, it comprises an antigen-binding portion of an antibody as described herein and typically contains a variable domain of the antibody. The variable domain of the functional portion may be a single-chain Fv fragment or a so-called single-domain antibody fragment. Preferred antibody portions or derivatives have at least two variable domains of an antibody or an equivalent thereof. Non-limiting examples of such variable domains or equivalents thereof are F(ab) fragments and single-chain Fv fragments. The functional portion of a bispecific antibody comprises an antigen-binding portion of the bispecific antibody or a derivative and/or analog of a binding portion. As mentioned above in the present specification, the binding portion of the antibody is encompassed by the variable domain.

Pharmaceutical compositions and administration

Also provided are pharmaceutical compositions comprising an antibody or functional portion, derivative and/or analog thereof of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 as disclosed herein and a pharmaceutically acceptable carrier. In certain aspects, the present disclosure provides a pharmaceutical composition comprising an EGFR/LGR5 bispecific antibody and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. In certain aspects, the present disclosure provides combinations or kit parts of a pharmaceutical composition comprising an EGFR/LGR5 bispecific antibody and separate pharmaceutical compositions comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

As used herein, the term "pharmaceutically acceptable" means approved by a governmental regulatory agency for use in animals, particularly humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia, and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or saline solutions and aqueous dextrose and glycerol solutions can be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration, including injection or infusion, include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the active compound may be coated with a substance to protect it from the action of acids and other natural conditions that may inactivate the compound.

The pharmaceutical composition comprising the antibody, functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR may be contained in a holder that is separate from, i.e., not physically connected to, the holder containing the pharmaceutical composition comprising the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or are suitable for reconstitution into liquid solutions or suspensions for intravenous administration. The compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Also included are solid preparations intended to be converted to liquid preparations for oral or parenteral administration immediately prior to use. Such liquid forms include solutions, suspensions, and emulsions.

The compositions and methods provided herein are particularly useful for the treatment of cancer in patients, particularly head and neck cancer, stomach cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer. Accordingly, the compositions and methods may be used for the treatment of various malignancies.

In certain aspects, the subject or patient is a mammal, particularly a human.

Previous treatment

In certain embodiments, the subject has not previously been treated with anticancer therapy. In other words, the subject's cancer has not previously been treated with anticancer therapy. In certain embodiments, the subject is anticancer treatment-naive. In certain embodiments, treatment with the therapeutic agent of the present disclosure is a first-line treatment. In certain embodiments, the subject has not previously been treated with neoadjuvant therapy. In certain embodiments, the cancer is head and neck cancer, stomach cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer.

In certain aspects, the subject has previously been treated with anticancer therapy. In other words, the subject's cancer has previously been treated with anticancer therapy. In certain aspects, the subject has previously received treatment with anticancer therapy. In other words, the subject's cancer has previously been treated with anticancer therapy.

In certain embodiments, the subject has previously been treated with two or more anticancer therapies. In other words, the subject's cancer has previously been treated with two or more anticancer therapies. In certain embodiments, the subject has previously received treatment with two or more anticancer therapies. In other words, the subject's cancer has previously been treated with two or more anticancer therapies.

In certain instances, the cancer is head and neck cancer, stomach cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer. In certain instances, the subject has previously been treated with neoadjuvant therapy.

In certain aspects, anticancer treatments include chemotherapy, radiation therapy, targeted therapy, and/or immunotherapy.

The subject may have previously been treated with one or more standard approved therapies or standard treatments. Surgery or radiation therapy may be preferred for most patients with early or localized disease, and may also be considered for locally advanced disease, but this may not be applicable to all patients, for example, due to the anatomic location of the cancer. In certain aspects, standard approved therapies or standard treatments herein include treatment by administration of a chemotherapeutic agent, such as one or more of a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, oxaliplatin), an antineoplastic compound (e.g., methotrexate), a fluoropyrimidine (e.g., fluorouracil, 5-FU, capecitabine), a taxane (e.g., docetaxel or paclitaxel), a nucleoside analog (e.g., gemcitabine), a BCL-2 inhibitor, or SN-38, or any combination thereof. In certain instances, chemotherapy is fluorouracil, TAS-102, oxaliplatin, venetoclax, SN-38, FOLFOX, or FOLFIRI.

Targeted therapies, as disclosed herein, include treatments using agents that block the action of specific enzymes, proteins, or other molecules involved in the growth or spread of cancer cells. Targeted therapies include, but are not limited to, angiogenesis inhibitors (e.g., bevacizumab) or monoclonal antibodies (e.g., trastuzumab, cetuximab, afatinib). In certain embodiments, the targeted therapy is cetuximab.

In certain embodiments, immunotherapy comprises treatment with an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor of the present disclosure targets an immune checkpoint protein selected from PD-L1, PD-1, CTLA-4, B7-1, or B7-2. In certain embodiments, the immune checkpoint inhibitor comprises durvalumab, pembrolizumab, ipilimumab, nivolumab, atezolizumab, retipanlimab, cemiplimab, or other approved or under development anti-PD1 or anti-PD-L1 antibodies. In certain embodiments, the immune checkpoint inhibitor comprises durvalumab or pembrolizumab.

In certain embodiments, the subject to be treated has progressed following treatment with fluorouracil, oxaliplatin, FOLFOX, FOLFIRI, targeted therapy, immunotherapy, or a combination thereof. In certain embodiments, the treatment is for colorectal cancer.

In certain embodiments, the subject or cancer to be treated has previously been treated with a first-line standard therapy, e.g., for CRC or mCRC. In certain embodiments, the subject or cancer to be treated has previously been treated with a second-line therapy comprising an antibody or functional portion thereof, derivative and/or analog comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5, in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, and such second-line therapy is administered.

In certain embodiments, the subject or cancer to be treated has been previously treated with a third-line therapy or a subsequent therapy using an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5 in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, and such third-line therapy or subsequent therapy is administered. In certain embodiments, the prior therapy comprises an appropriate standard therapy for CRC or mCRC. In certain embodiments, the standard therapy comprises or is an appropriate chemotherapy for CRC or mCRC. In certain embodiments, the prior therapy comprises or is an appropriate chemotherapy for CRC or mCRC. In certain embodiments, the prior therapy comprises or is an appropriate fluoropyrimidine-oxaliplatin-based chemotherapy with or without bevacizumab or a fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab, appropriate for CRC or mCRC.

In certain aspects, the subject or cancer has been previously treated with a fluoropyrimidine-oxaliplatin-based chemotherapy, with or without bevacizumab, and the antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5 is administered, and FOLFIRI is administered. In certain aspects, the subject or cancer has been previously treated only with the oxaliplatin-based chemotherapy. The administration of the antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5 and FOLFIRI is provided as a second-line therapy.

In certain embodiments, the subject or cancer has been previously treated with fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab and is administered an antibody or functional portion, derivative and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5, and FOLFOX is administered.

In certain embodiments, the subject or cancer has previously been treated solely with irinotecan-based chemotherapy. Administration of the antibody or functional portion, derivative, and/or analog thereof comprising a variable domain that binds the extracellular portion of EGFR and optionally a variable domain that binds the extracellular portion of LGR5, and FOLFIRI, is provided as a second-line therapy.

In certain embodiments, the subject to be treated has progressed following treatment with fluorouracil, oxaliplatin, FOLFOX, FOLFIRI, targeted therapy, immunotherapy, or a combination thereof. In certain embodiments, the treatment is for gastric cancer.

In certain embodiments, the subject to be treated has progressed following treatment with a fluoropyrimidine, oxaliplatin, FOLFOX, FOLFIRI, targeted therapy, immunotherapy, or a combination thereof. In certain embodiments, the treatment is for esophageal or gastroesophageal junction cancer.

In certain embodiments, the subject to be treated has progressed following treatment with radiation therapy, fluoropyrimidine, platinum-based therapy, targeted therapy, immunotherapy, or a combination thereof. In certain embodiments, the treatment is for head and neck cancer.

In certain instances, the subject being treated has progressed following treatment with platinum-based therapy, targeted therapy, immunotherapy, or a combination thereof. In certain instances, the treatment is for non-small cell lung cancer.

administration

Through its Biclonics® antibody program, Merus has developed multispecific antibodies targeting EGFR and LGR5 (a leucine-rich repeat-containing G protein-coupled receptor). The efficacy of these multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, e.g., WO2017/069628, incorporated herein by reference). The multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of these inhibitory antibodies has been shown to correlate with the level of LGR5 RNA expression by cancer cells. In certain aspects, the multispecific antibodies targeting EGFR and LGR5 are as described in WO2017/069628.

The present disclosure further provides a method of treating such cancer in a subject, comprising administering to the subject in need thereof an antibody or a functional portion, derivative, and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38. In certain aspects, the method comprises providing the subject with a dose of 1500 mg of the antibody or a functional portion, derivative, and/or analog thereof. In certain aspects, the method comprises providing the subject with a flat dose of 1500 mg of the antibody or a functional portion, derivative, and/or analog thereof. In certain aspects, administration of the combination of therapeutic agents or agents to the subject can be performed weekly, every other week, or monthly. In certain aspects, the combination of therapeutic agents or agents is administered every two weeks. In certain aspects, the therapeutic agent is fetosemtamab and is administered at 1500 mg every two weeks.

In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of 5 to 2000 mg. In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of 5, 20, 50, 90, 150, 225, 335, 500, 750, 1100, 1500 mg or 2000 mg. In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of 750, 1100, 1500 mg or 2000 mg. In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of 750 mg. In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of 1100 mg. In certain aspects, the administration of the antibody or functional part, derivative and/or analog thereof is performed in a dosage of Administration of the antibody or functional portion, derivative and/or analog thereof is performed in a dosage of 1500 mg. In certain aspects, administration of the antibody or functional portion, derivative and/or analog thereof is performed in a dosage of 2000 mg. As will be understood by those skilled in the art, the dosage may be administered over time. In certain aspects, the dosage is administered by IV, for example, as a 1 to 6 hour infusion or a 2 to 4 hour infusion. In certain aspects, the dosage is administered once every two weeks. In certain aspects, the dosage is a flat dosage suitable for use in adults and/or subjects weighing at least 35 kg.

In certain embodiments, petosemtamab is administered in a dosage of 5 to 2000 mg. In certain embodiments, petosemtamab is administered in a dosage of 5, 20, 50, 90, 150, 225, 335, 500, 750, 1100, 1500 mg, or 2000 mg. In certain embodiments, petosemtamab is administered in a dosage of 750 mg. In certain embodiments, petosemtamab is administered in a dosage of 1100 mg. In certain embodiments, petosemtamab is administered in a dosage of 1500 mg. In certain embodiments, petosemtamab is administered in a dosage of 2000 mg. As will be understood by those skilled in the art, the dosages may be administered over time. In certain embodiments, the dosage is administered intravenously, for example, as a 1-6 hour infusion or a 2-4 hour infusion. In certain embodiments, the dosage is administered once every two weeks. In certain embodiments, the dosage is a uniform dosage suitable for use in adults and/or subjects weighing at least 35 kg.

In certain embodiments, petosemtamab is administered as a weekly dose of 750 mg. In certain embodiments, petosemtamab is administered as a biweekly dose of 750 mg. In certain embodiments, petosemtamab is administered as a triweekly dose of 750 mg.

In certain embodiments, petosemtamab is administered as a weekly dose of 1100 mg. In certain embodiments, petosemtamab is administered as a biweekly dose of 1100 mg. In certain embodiments, petosemtamab is administered as a triweekly dose of 1100 mg.

In certain embodiments, petosemtamab is administered as a weekly dose of 1500 mg. In certain embodiments, petosemtamab is administered as a biweekly dose of 1500 mg. In certain embodiments, petosemtamab is administered as a triweekly dose of 1500 mg.

In certain embodiments, the antibody or functional portion, derivative and/or analog thereof comprises petosemtamab and is administered in an amount that achieves at least 90%, at least 95%, or at least 99% human receptor target engagement for both EGFR and LGR5 across a statistically significant number of subjects over a relevant body weight. The 90% amount can be achieved using a flat dose of about 1000 mg Q2W. The 95% amount can be achieved using a flat dose of about 1100 mg to about 1200 mg Q2W.

In certain embodiments, premedication therapy is used. Such therapy may be applied to reduce the likelihood or severity of infusion-related reactions. Typically, a steroid, such as dexamethasone, and/or an antihistamine, such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine, are administered (e.g., orally, intravenously) prior to treatment with the therapeutic agents described herein. In certain embodiments, the treatment of the present disclosure includes premedication with paracetamol/acetaminophen, an antihistamine, or a corticosteroid. In certain embodiments, the premedication is administered in accordance with standard local clinical practice if infusion-related reactions, hypersensitivity, and/or allergic reactions occur. In certain embodiments, the 1500 mg dose of fetosemtamab is premedicated with an antihistamine, an analgesic, an antipyretic, and/or an anti-inflammatory agent. In certain instances, the 750 mg dose of petosemtamab is premedicated with antihistamines, analgesics, antipyretics, and/or anti-inflammatory agents.

As used herein, combination therapy, administration or co-administration includes simultaneous treatment or administration of an antibody or functional portion, derivative and/or analog thereof of the present disclosure with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the same or different dosage forms, or separately or sequentially. Thus, in certain aspects, the antibody or functional portion, derivative and/or analog thereof can be used in a method of treating cancer in a subject, wherein the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, separately or sequentially with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. In another aspect, the antibody or functional part, derivative and/or analog thereof can be used for the treatment of cancer in a subject, wherein the antibody or functional part, derivative and/or analog thereof can be administered simultaneously, separately or sequentially with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In some aspects, the antibody or functional part, derivative and/or analog thereof may be for use in the manufacture of a medicament for treating cancer in a subject, wherein the antibody or functional part, derivative and/or analog thereof is administered simultaneously, separately or sequentially with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. In some aspects, the antibody or functional part, derivative and/or analog thereof may be for use in the manufacture of a medicament for treating cancer in a subject, wherein the antibody or functional part, derivative and/or analog thereof may be administered simultaneously, separately or sequentially with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

In some embodiments, the antibody or functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 may be used in the manufacture of one or more medicaments for treating cancer in a subject, wherein the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, separately or sequentially with the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

The product comprising the antibody or a functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 may be a combination formulation for use simultaneously, separately or sequentially to treat cancer in a subject.

The antibody or functional portion, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may also be administered according to any suitable schedule. In certain aspects, the antibody or functional portion, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be administered simultaneously in a single formulation. In certain aspects, the antibody or functional portion, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be formulated for separate administration, and they are administered simultaneously or sequentially.

For example, in certain embodiments, the antibody or functional portion, derivative, and/or analog thereof may be administered first, followed by the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor, or SN-38. Dosage regimens in the above methods of treatment and use are adjusted to provide the optimal desired response (e.g., therapeutic response).

The antibody or functional portion thereof, derivative and/or analog disclosed herein and a fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be administered at a suitable dosage and via a suitable route (e.g., intravenously, intraperitoneally, intramuscularly, intrathecally or subcutaneously). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

In certain aspects, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of the antibody or functional portion, derivative and/or analog thereof and an effective amount of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

In certain aspects, the antibody or functional portion, derivative and/or analog thereof is administered prior to administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38, e.g., the antibody or functional portion, derivative and/or analog thereof is administered to the patient first, followed by administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38. In one aspect, the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38 is administered prior to administration of the antibody or functional portion, derivative and/or analog thereof, e.g., the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38 is administered to the patient first, followed by administration of the antibody or functional portion, derivative and/or analog thereof (e.g., at least 1 minute, at least 1 hour or at least 1 day later). Such simultaneous or sequential administration results in the simultaneous presence of both the antibody or functional part, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 in the treated patient. The simultaneous presence of both the antibody or functional part, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 will support the cancer treatment induced by the antibody or functional part, derivative and/or analog thereof and the inhibition of EGFR/LGR5 signaling mediated by the antibody or functional part, derivative and/or analog thereof.

In certain embodiments, the subject is administered a single dose of an antibody or functional portion, derivative, and/or analog thereof as disclosed herein and a single dose of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38. In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor, or SN-38 are administered repeatedly over the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the antibody or functional portion, derivative and/or analog thereof are administered to a subject in need thereof.

In certain embodiments, the administration of the antibody or functional portion, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be performed weekly, biweekly or monthly, and in the regimen, they may be administered on the same day (e.g., simultaneously) or one after the other (e.g., at least 1 minute, at least 1 hour or at least 1 day before or after each other). When administered separately, the antibody or functional portion, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may, but need not, be administered according to the same administration (i.e., dosing) protocol. For example, a treatment cycle may include one or more administrations of the antibody or functional portion, derivative and/or analog thereof, while a therapeutically effective dose of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be administered more or less frequently than the antibody or functional portion, derivative and/or analog thereof. In certain aspects, administration of each dose of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and the antibody or functional portion, derivative and/or analog thereof may occur on the same day, or alternatively, the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be administered one or more days before or after administration of the antibody or functional portion, derivative and/or analog thereof.

In certain embodiments, the dose of the antibody or functional portion, derivative and/or analog thereof and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 is varied over time. For example, the antibody or functional portion, derivative and/or analog thereof and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 may be administered initially at a high dose and then lowered over time. In another embodiment, the antibody or functional portion, derivative and/or analog thereof and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 is administered initially at a low dose and then increased over time.

In certain embodiments, the amount of the antibody or functional portion thereof, derivative and/or analog and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 is constant for each dose. In other embodiments, the amount of the antibody or functional portion thereof, derivative and/or analog and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 is varied for each dose. For example, each maintenance (or subsequent) dose may be higher than or equal to the initially administered loading dose. In other embodiments, each maintenance dose may be lower than or equal to the loading dose. The clinician may use a preferred dosage that warrants treatment of the patient's condition. The dosage may vary depending on several factors, including the stage of the disease. It is within the skill of the art to determine the specific dosage to be administered based on the presence of one or more of these factors. Typically, treatment is initiated at a dose lower than the optimal dose of the compound. Thereafter, the dosage is increased in small increments, depending on the situation, until the optimal effect is achieved. For convenience, if desired, the total daily dose may be divided and administered throughout the day. Intermittent administration (e.g., once every three weeks or three out of four weeks) may also be used.

In certain embodiments, the antibody or functional portion, derivative and/or analog thereof is administered at a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg body weight. Alternatively, the antibody or functional portion, derivative and/or analog thereof is administered at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg body weight. In certain embodiments, the antibody or functional portion, derivative and/or analog thereof is administered to the subject using a 1500 mg flat dose. Flat dose administration provides several advantages over body surface or body weight dosing because it reduces manufacturing time and potential dose calculation errors. In certain embodiments, the antibody or functional portion, derivative and/or analog thereof is administered at a dose of at least 500 mg. In certain embodiments, the dosage is 1100 to 2000 mg. In certain embodiments, the dosage is 1100 to 1800 mg. As will be understood by those skilled in the art, the dosage may be administered over time. For example, the dosage may be administered by IV, for example, as a 1 to 6 hour infusion or a 2 to 4 hour infusion. In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof is administered once every two weeks. In certain embodiments, the antibody is petosemtamab and is administered as a 1500 mg flat dose once every two weeks. In particular, the flat doses disclosed herein are suitable for use in adults and/or subjects weighing at least 35 kg. In certain embodiments, the subject suffers from head and neck cancer, stomach, esophagus, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer.

In certain instances, premedication may be used. This therapy may be used to reduce the likelihood or severity of infusion-related reactions. Typically, a steroid, such as dexamethasone, and/or an antihistamine, such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine, are administered (e.g., orally or intravenously) prior to antibody therapy.

The treatment methods described herein are typically continued for as long as the clinician supervising the patient's treatment determines that the treatment is effective, i.e., as long as the patient responds to the treatment. Non-limiting parameters indicating that the treatment is effective may include one or more of the following: reduction of tumor cells; inhibition of tumor cell proliferation; elimination of tumor cells; progression-free survival; and adequate response as determined by appropriate tumor markers (if applicable).

With respect to the frequency of administration of the antibody or functional portion thereof, derivative and/or analog and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38, one of ordinary skill in the art will be able to determine an appropriate frequency. For example, the clinician may decide to administer the antibody or functional portion thereof, derivative and/or analog and/or fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 relatively infrequently (e.g., once every two weeks) and gradually shorten the period between doses as tolerated by the patient. Exemplary time periods associated with a course of therapy according to the claimed methods include: about 1 week; 2 weeks; about 3 weeks; about 4 weeks; about 5 weeks; about 6 weeks; about 7 weeks; about 8 weeks; about 9 weeks; about 10 weeks; about 11 weeks; about 12 weeks; about 13 weeks; about 14 weeks; About 15 weeks; About 16 weeks; About 17 weeks; About 18 weeks; About 19 weeks; About 20 weeks; About 21 weeks; About 22 weeks; About 23 weeks; About 24 weeks; About 7 months; About 8 months; About 9 months; About 10 months; About 11 months; About 12 months; About 13 months; About 14 months; About 15 months; About 16 months; About 17 months; About 18 months; About 19 months; About 20 months; About 21 months; About 22 months; About 23 months; About 24 months; About 30 months; About 3 years; About 4 years; About 5 years; Permanent (e.g., continuous maintenance therapy). The aforementioned durations may relate to one or multiple treatment sessions/cycles.

The efficacy of the treatment methods provided herein can be assessed using any suitable means. In certain embodiments, the clinical efficacy of the treatment is assessed using a reduction in cancer cell count as an objective response criterion. Patients, e.g., humans, treated according to the methods disclosed herein preferably experience an improvement in at least one symptom of their cancer. In certain embodiments, one or more of the following may occur: a decrease in cancer cell count; a prevention or delay in cancer recurrence; or some alleviation of one or more symptoms associated with the cancer. Additionally, in vitro assays are used to determine T cell-mediated target cell lysis. In certain embodiments, tumor evaluation is based on CT scans and/or MRI scans, see, e.g., RECIST 1.1 (Response Evaluation Criteria in Solid Tumors) guidelines (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). These evaluations are typically performed every 4 to 8 weeks after treatment.

In certain embodiments, tumor cells are no longer detectable after treatment as described herein. In certain embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has increased overall survival, median survival, and/or progression-free survival.

The combination of the present disclosure (i.e., the antibody or functional portion, derivative and/or analog thereof in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38) may also be used in combination with other well-known therapies (e.g., chemotherapy or radiotherapy) selected for their particular utility in the cancer being treated.

Safe and effective methods for administering chemotherapeutic agents are well known to those skilled in the art. Furthermore, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA); the disclosures of which are incorporated herein by reference.

It will be apparent to those skilled in the art that the administration of chemotherapeutic agents and/or radiation therapy may vary depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease. Furthermore, within the knowledge of a skilled clinician, treatment protocols (e.g., dosage and frequency of administration) may vary based on the observed effects of the administered treatment on the patient and the observed response of the disease to the administered treatment.

In certain aspects, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1 through IF16 are applicable to the subject being treated. In certain aspects, the subject includes or complies with all of IF1 through IF16:

IF 1. You are at least 18 years old.

IF 2. There is a histologically or cytologically confirmed solid tumor with evidence of metastatic or locally advanced disease that is not amenable to standard treatment with curative intent, or locally advanced unresectable or metastatic disease.

IF 3. Previous diagnosis of unresectable or metastatic colon or rectal adenocarcinoma, confirmed histologically or cytologically. Subject is RAS/RAF wild-type as determined using NGS on tumor tissue (primary or metastatic).

F4. The subject is naive to previous anti-EGFR therapy.

IF5. Disease progression must have occurred during or within 6 months of receiving the previous first-line chemotherapy as provided and must have been confirmed by appropriate radiological examination.

IF6.1. Subjects receiving treatment with the combination of petosemtamab and FOLFIRI must have received a single prior chemotherapy regimen for metastatic disease, typically first-line fluoropyrimidine-oxaliplatin-based chemotherapy ± bevacizumab.

IF6.2. Subjects receiving treatment with the combination of petosemtamab and FOLFOX must have received a single prior chemotherapy regimen for metastatic disease, typically first-line fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab.

IF 7. A new baseline tumor sample (e.g., foam-fixed, paraffin-embedded block [FFPE]) is obtained from a metastatic or primary site. If the subject has a tumor sample available from a collection with sufficient material (at least 20 slides with >20% tumor content) and has not received any additional anticancer treatment since sample collection, a new tumor biopsy is not required at baseline. Archival FFPE slides are not acceptable.

IF 8. Suitable for biopsy.

IF 9. Measurable disease as defined by RECIST version 1.1 by radiological methods.

Eastern Cooperative Oncology Group (ECOG) performance status of IF 10.0 or 1.

IF 11. According to the investigator, life expectancy is 12 weeks or more.

IF 12. Left ventricular ejection fraction (LVEF) of at least 50% by echocardiography (ECHO) or multi-gated acquisition scanning (MUGA).

IF 13. Appropriate organ function:

IF 13.1 Absolute neutrophil count (ANC) at least 1.5× ¹⁰⁹ /L.

IF 13.2 Hemoglobin level at least 9 g/dL.

IF 13.3 Platelet level at least 100×10 ⁹ /L.

IF 13.4 Corrected total serum calcium within normal range.

IF 13.5 Serum magnesium, sodium, corrected total calcium, phosphate, and potassium within normal ranges (or corrected by supplements or appropriate treatment).

IF 13.6 Alanine aminotransferase (ALT), aspartate aminotransferase (AST) 2.5 × the upper limit of normal (ULN) and total bilirubin 1.5 × ULN or less, except if the subject has Gilbert syndrome, total bilirubin 3.0 × ULN or less or direct bilirubin 1.5 × ULN or less; except in liver involvement, ALT/AST 5 × ULN or less and total bilirubin 2 × ULN or less.

IF 13.7 Serum creatinine level 1.5×ULN or less or creatinine clearance of at least 60 mL/min as calculated according to the Cockroft and Gault formula or, for patients over 65 years of age, the Modification of Diet in Renal Disease (MDRD) formula.

IF 13.8 Serum albumin level at least 3 g/dL.

IF 13.9 International normalized ratio (INR) or prothrombin time (PT) level ≤ 1.5 × ULN, unless the patient is receiving anticoagulant therapy and is within the therapeutic range of the anticoagulant used intentionally.

IF 13.10 Activated partial thromboplastin time (APTT) or PTT 1.5×ULN or less, unless the patient is receiving anticoagulant therapy and is within the therapeutic range of the anticoagulant used intentionally.

IF 14. Subjects are willing to be tested for human immunodeficiency virus (HIV) if they have not been tested within the past 6 months. Known HIV-positive subjects are eligible if they have a CD4+ count ≥300/μL, a undetectable viral load, and are currently receiving highly active antiretroviral therapy (HAART).

IF 15. Hepatitis B (HBsAg) positive, but receiving antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents, starting at least 7 days prior to starting treatment according to this disclosure.

IF 16. There is a history of hepatitis B (anti-HBc positive, HBsAg and hepatitis B virus (HBV) DNA negative).

IF 17. A positive test result for hepatitis C virus (HCV) ribonucleic acid (RNA), provided that the HCV infection has spontaneously resolved (i.e., HCV antibody is positive but HCV RNA is undetectable) or the subject has achieved a sustained response to antiviral treatment and has demonstrated the absence of detectable HCV RNA for at least 6 months using an IFN-free regimen or at least 12 months using an IFN-based regimen after discontinuation of antiviral treatment.

In certain aspects, all organ function measures according to IF13 have upper limits observed in healthy subjects.

In certain aspects, the subject of treatment complies with any one or more elements selected from the group consisting of IF1 to IF16. In certain aspects, the subject of treatment complies with elements IF2, IF3, IF4, IF5, IF6, IF9, IF10, IF12, IF13, IF14, IF15, IF16, and IF17. In certain aspects, the subject of treatment complies with elements IF2, IF3, IF4, IF5, and IF6. In certain aspects, the subject of treatment complies with elements IF3 and IF6.

In certain aspects, at the start of treatment, at least one, more than one, or all of the following exclusion factors EF1 to EF18 are applicable to the subject being treated.

EF 1. Central nervous system metastases that are unresponsive, symptomatic, or require radiation therapy, surgery, or ongoing steroid therapy within 14 days of starting treatment according to this disclosure.

EF 2. There is leptomeningeal invasion.

EF 3. Participation in additional clinical trials or treatment with any investigational drug within 4 weeks prior to starting treatment according to this disclosure.

EF 4. Any systemic anticancer treatment within 4 weeks or 5 half-lives (whichever is longer) of the first dose according to the present disclosure. For cytotoxic agents with major delayed toxicities (e.g., mitomycin C, nitrosoureas) or anticancer immunotherapy, a 6-week washout period is required prior to starting treatment according to the present disclosure.

EF 5. Requirement for immunosuppressive medications (e.g., methotrexate, cyclophosphamide).

EF 6. Major surgery or radiation therapy performed within 3 weeks of starting treatment according to this disclosure. Patients who have previously received radiation therapy to at least 25% of the bone marrow are excluded, regardless of the timing of treatment.

EF 7. Persistent Grade 1 or greater clinically significant toxicity (excluding alopecia) related to previous antineoplastic therapy; except for stable sensory neuropathy Grade 2 (or less) National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03 or v5.0 or criteria valid at the time of administration are acceptable.

EF 8. History of exposure to excipients of petosemtamab required for this study, human proteins, or non-IMP treatments.

EF 9. Indicates hypertension (systolic > 150 mmHg and/or diastolic > 100 mmHg) or unstable angina that is not controlled with appropriate treatment.

EF 10. Congestive heart failure of New York Heart Association (NYHA) class II to IV or history of serious cardiac arrhythmia requiring treatment (excluding atrial fibrillation or paroxysmal supraventricular tachycardia).

EF 11. History of myocardial infarction within 6 months of starting treatment according to this disclosure.

EF 12. History of previous malignancy other than resected cervical intraepithelial neoplasia or non-melanoma skin cancer, or cured cancer with no evidence of disease for at least 3 years prior to initiation of treatment according to the present disclosure and considered to be at low risk of recurrence.

EF 13. If you have dyspnea at rest of any origin or any other condition requiring continuous oxygen therapy.

EF 14. History of interstitial lung disease (ILD) (e.g., pneumonia or pulmonary fibrosis) or evidence of ILD on baseline chest computed tomography (CT) scan.

EF 15. Current serious illness or medical condition at the time of initiation of treatment according to this disclosure, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorders.

EF 16. Subjects with active hepatitis B surface antigen infection (HBsAg positive) who are not receiving antiviral treatment.

EF 17. Hepatitis C virus (HCV) test result is positive.

EF 18. Subjects with cirrhosis of Child-Pugh class B or C; subjects with fibrolamellar HCC, subjects with sarcomatoid HCC, or subjects with mixed cholangiocarcinoma and HCC.

EF 18. Pregnant or lactating subjects; Subjects of childbearing potential must use highly effective contraception before starting treatment according to the present disclosure, during said treatment, and for 6 months after the last dose of petosemtamab.

In certain embodiments, the subject of treatment complies with one or more elements selected from the group consisting of EF1 to EF18. In certain embodiments, the subject of treatment complies with all elements EF1 to EF18. In certain embodiments, the subject of treatment complies with elements IF3 and IF16.

The ECOG performance status score grades are defined as follows: 0 Fully active and able to perform all pre-disease activities without limitations. 1 Limited in physically strenuous activities but able to ambulate and perform light or sedentary tasks such as light housework or office work. 2 Ambulatory and able to perform all self-care but unable to perform any occupational activities. Active for approximately 50% or more of waking hours. 3 Limited self-care and confined to bed or chair for more than 50% of waking hours. 4 Totally disabled. Unable to perform any self-care. Entirely confined to bed or chair. 5 Dead.

The Child-Pugh score, also known as the Child-Pugh classification, Child-Turcotte-Pugh (CTP) calculator, or Child criteria, is used herein according to standard clinical practice. The Child-Pugh score determines the likelihood of liver failure by scoring five clinical measures of liver disease. Each measure is given a score of 1, 2, or 3, with 3 being the most severe. The five clinical measures are total bilirubin, serum albumin level, prothrombin time (or prolonged blood clotting time, or INR), ascites, and hepatic encephalopathy. Grade A, with a score of 5 to 6, indicates the least severe liver disease and a 95% chance of survival at 1 to 5 years. Grade B, with a score of 7 to 9, indicates moderately severe liver disease and a 75% chance of survival at 1 to 5 years. Grade C, with a score of 10 to 15, indicates the most severe liver disease and a 50% chance of survival at 1 to 5 years. The following scores are assigned according to the clinical scale: Encephalopathy: None = 1 point, Grades 1 and 2 = 2 points, Grades 3 and 4 = 3 points. Ascites: None = 1 point, mild = 2 points, moderate = 3 points. Bilirubin: Less than 2 mg/ml = 1 point, 2 to 3 mg/ml = 2 points, >3 mg/ml = 3 points. Albumin: More than 3.5 mg/ml = 1 point, 2.8 to 3.5 mg/ml = 2 points, <2.8 mg/ml = 3 points. Prothrombin time (PT, prolonged in seconds): Less than 4 seconds = 1 point, 4 to 6 seconds = 2 points, >6 seconds = 3 points. Alternatively, the international normalized ratio (INR) can be used instead of PT, with INR less than 1.7 = 1 point, INR 1.7 to 2.2 = 2 points, and INR greater than 2.2 = 3 points.

In this specification, renal clearance is calculated according to the Cockcroft and Gault formula for subjects younger than 65 years: Male = 1.25 × body weight (kg) × (140-age)/serum creatinine (μmol/L). Female = 1.04 × body weight (kg) × (140-age)/serum creatinine (μmol/L).

In this specification, the Modification of Dietary Ratings in Renal Disease (MDRD) is calculated according to the following formula: Patients over 65 years of age. For male subjects over 65 years of age, the calculation is: Male = 186 × (Serum Creatinine (μmol/L) × 0.0113) - 1.154 × Age - 0.203. For subjects with dark skin, multiply the result by 1.21. For female subjects, multiply the result by 0.742.

Combination of kit parts and remedies

In certain aspects, the present disclosure provides a kit or article comprising a pharmaceutical composition comprising a therapeutically effective amount of an EGFR/LGR5 bispecific antibody configured for use in the preceding method and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38, and a pharmaceutically acceptable carrier. In certain aspects, the kit or article may optionally also include instructions, including, for example, a dosing schedule, by which a healthcare professional (e.g., a physician, nurse, or patient) can administer the composition included in the kit or article to a cancer patient.

Accordingly, in certain aspects, the present disclosure provides a kit component comprising:

- an antibody or a functional part, derivative and/or analog thereof,

- Fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38; and

- Instructions for use of EGFR/LGR5 bispecific antibodies and instructions for use of fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38.

In certain embodiments, the instructions for use of petosemtamab include instructions for a 1500 mg dose. In certain embodiments, the instructions for use are for the use of the antibody or a functional portion, derivative, and/or analog thereof, a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 in the treatment of adenocarcinoma or squamous cell carcinoma. In certain embodiments, the instructions for use are for the treatment of head and neck cancer, stomach, esophageal, gastroesophageal junction cancer, non-small cell lung cancer, or colorectal cancer. In certain embodiments, the instructions for use are for the treatment of colorectal cancer.

In certain aspects, the kit or article comprises a plurality of packages of single-dose pharmaceutical compositions each containing an effective amount of the antibody or functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for a single administration according to the methods provided above. Devices or apparatus necessary for administering the pharmaceutical composition(s) may also be included in the kit or article. For example, the kit or article may provide one or more pre-filled syringes containing unit doses of the antibody or functional portion, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the same container or separate containers to be administered as separate and distinct compositions.

In certain embodiments, the antibody or functional portion, derivative and/or analog thereof and one or both of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 are provided in a solid form suitable for reconstitution and subsequent administration according to the attached instructions.

In certain aspects, the present disclosure provides a combination of an antibody or a functional portion, derivative and/or analog thereof with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for use in the treatment of cancer in a subject in need thereof.

In certain aspects, the present disclosure provides a combination of:

- an antibody of the present disclosure or a functional part, derivative and/or analog thereof,

- Instructions for use of the antibody or its functional part, derivative and/or analogue in the treatment of cancer in a subject;

- Instructions for use in the treatment of cancer in subjects receiving fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38.

In certain aspects, the present disclosure provides a combination of:

- Fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38; and

- Instructions for use of fluoropyrimidines, platinum-based chemotherapeutics, BCL-2 inhibitors or SN-38 in the treatment of cancer in subjects;

- Instructions for use of the antibody or a functional part, derivative and/or analog thereof in the treatment of cancer in a subject.

In certain embodiments, the instructions for use include the amount of the antibody or functional portion, derivative and/or analog thereof to be used and/or the amount and/or dosing interval of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and/or the cancer to be treated.

In a further aspect, the composition or combination or kit or product comprises one or more additional active agents.

The compounds and compositions disclosed herein are useful as therapies and in therapeutic treatments, and thus may be useful as medicaments and may be used in methods of making medicaments.

In certain aspects, the present disclosure provides a pharmaceutical composition comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and instructions for use thereof with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of said cancer.

In certain aspects, the present disclosure provides a pharmaceutical composition for treating cancer comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and a pharmaceutical composition for treating cancer comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 of the present disclosure.

In certain aspects, the present disclosure provides a pharmaceutical composition for use in the treatment of cancer comprising an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, wherein the pharmaceutical composition is administered in combination with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 of the present disclosure.

In certain aspects, the present disclosure relates to a pharmaceutical composition for the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional portion, derivative and/or analog thereof, wherein the subject to be treated is administered a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, either prior to or concurrently with administration of the bispecific antibody.

In certain aspects, the present disclosure relates to a pharmaceutical composition for treating cancer in a subject comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38, wherein the subject to be treated is administered an antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure prior to, concurrently with, or subsequent to administration of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor, or SN-38.

Accordingly, the present disclosure relates to a combination of medicaments for treating cancer in a subject, comprising administering to the subject a plurality of different medicaments for treating said cancer, wherein the treatment comprises simultaneous, sequential, or separate administration of the medicaments. In certain aspects, the medicament comprises an antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, and the other different medicaments comprise a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38.

In certain embodiments, the antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure may be administered simultaneously, sequentially, or separately with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 of the present disclosure. Thus, the combination of the antibody or functional portion, derivative, and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor, or SN-38 encompasses simultaneous, sequential, or separate administration.

Accordingly, in certain aspects, the present disclosure provides an antibody or functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure for use in a method of treating cancer, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, optionally wherein the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

Accordingly, in certain aspects, the present disclosure provides a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 and an antibody or functional portion, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, wherein optionally the antibody or functional portion, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38.

Accordingly, in certain aspects, the present disclosure provides the use of an antibody or a functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the manufacture of a medicament for the treatment of cancer, wherein optionally the antibody or a functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38. In certain aspects, the antibody or a functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or the SN-38.

In certain aspects, an antibody or a functional part, derivative and/or analog thereof comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure is for use in the manufacture of a medicament for treating cancer, wherein a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 is for use in the manufacture of a medicament for treating said cancer, wherein optionally the antibody or a functional part, derivative and/or analog thereof is administered simultaneously, sequentially or separately from the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38. Optionally, the antibody or a functional part, derivative and/or analog thereof is administered before, simultaneously or after the administration of the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.

definition

To facilitate a better understanding of this description, certain terms are defined herein as follows. Additional definitions may be provided throughout the detailed description, where deemed necessary.

As used herein, the singular form includes plural referents. The use of the terms "including," "having," and "including," as well as other forms such as "includes," "included," "has," "has," and "had," is not limiting.

The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins containing one or more domains that bind to an epitope on an antigen, wherein such domains are, are derived from, or share sequence homology with the variable region of an antibody. Antibodies are typically composed of basic structural units, each of which has two heavy chains and two light chains. The antibodies according to the present invention are not limited to any particular format or method of production thereof.

A "bispecific antibody" is an antibody described herein, wherein one domain of the antibody binds to a first antigen, while a second domain of the antibody binds to a second antigen, wherein the first and second antigens are not identical, or wherein one domain binds to a first epitope on the antigen, while the second domain binds to a second epitope on the antigen. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region/light chain variable region (VH/VL) combination binds to a first antigen or epitope on the antigen, and a second VH/VL combination binds to a second antigen or epitope on the antigen. The term further encompasses antibodies in which the VH is capable of specifically recognizing a first antigen, and the VL, paired with the VH in the immunoglobulin variable region, is capable of specifically recognizing a second antigen. The resulting VH/VL pair will bind to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO 2010/108127 and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of producing them.

The term "common light chain" as used herein refers to two light chains (or VL portions thereof) in a bispecific antibody. The two light chains (or VL portions thereof) may be identical or may have some amino acid sequence differences that do not affect the binding specificity of the full-length antibody. The terms "common light chain," "common VL," "single light chain," and "single VL," with or without the term "rearranged," are all used interchangeably herein. "Common" also refers to a functional equivalent of a light chain that does not have the same amino acid sequence. Many variants of the light chain exist, which include mutations (deletions, substitutions, insertions, and/or additions) that do not affect the formation of a functional binding domain. The light chain of the present disclosure may also be a light chain as specified herein, having 0 to 10 amino acid insertions, deletions, substitutions, additions, or a combination thereof. The light chain of the present disclosure may also be a light chain as described herein, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or any combination thereof. For example, it is within the scope of the definition of a common light chain as used herein to create or discover a non-identical, but still functionally equivalent, light chain by introducing and testing conservative amino acid changes, amino acid changes in regions that do not or only partially contribute to binding specificity when paired with a heavy chain, and the like.

As used herein, the words "comprising" and its conjugations are used in a non-limiting sense, meaning that the item following the word is included, but not excluding items not specifically mentioned. Additionally, the verb "consisting of" may be replaced with "consisting essentially of," meaning that a compound or adjunct compound as defined herein may include additional component(s) other than those specifically identified, provided that said additional component(s) do not alter the inherent characteristics of the invention.

An "antibody derivative" is a protein that deviates from the amino acid sequence of a native antibody by up to 20 amino acids in the CDR region. An antibody derivative as disclosed herein is an antibody that deviates from said amino acid sequence by up to 20 amino acids. The functional portion, derivative, and/or analog retains the binding specificity of the (bispecific) antibody. An "antibody analog" is a protein that may differ in structure, conformation, or origin, but retains the binding specificity of the antibody to which it is an analog.

When referring to nucleic acid or amino acid sequences herein, "percent identity (%)" is defined as the percentage of residues in a candidate sequence that are identical with the residues in the selected sequence after aligning the sequences for optimal comparison purposes. Percent sequence identity comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software using default settings, which uses a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J., (1994) Nuc. Acid Res. 22(22): 4673-4680), a swgapdnamt score matrix, a gap opening penalty of 15, and a gap extension penalty of 6.66. Amino acid sequences were aligned with the AlignX application in Vector NTI Advance® 11.5.2 software using default settings, which uses a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J., (1994) Nuc. Acid Res. 22(22): 4673-4680), blosum62mt2 score matrix, gap opening penalty of 10, and gap extension penalty of 0.1.

Since antibodies typically recognize epitopes on antigens, and these epitopes may also be present on other compounds, an antibody according to the invention that "specifically recognizes" an antigen, for example EGFR or LGR5, may also recognize other compounds if these other compounds contain the same type of epitope. Thus, the term "specifically recognizes" in relation to antigen and antibody interactions does not exclude binding of the antibody to other compounds containing the same type of epitope.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both consecutive amino acids or discontinuous amino acids juxtaposed by tertiary folding of the protein (so-called linear and conformational epitopes). Epitopes formed from consecutive linear amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding and conformation are typically lost upon treatment with denaturing solvents. Epitopes can typically comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. (e.g., a patient, such as a human patient having cancer).

In this specification, the words cancer and tumor are used, and unless specifically stated otherwise, both words generally refer to cancer.

The terms "treat," "treating," and "treatment" as used herein refer to any type of intervention or process performed on a subject or the administration of an active agent or combination of active agents to said subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the progression, onset, aggravation, or recurrence of a symptom, complication, condition, or biochemical sign associated with a disease.

As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, such as an improvement in at least one symptom of a disease or disorder, such as cancer. The beneficial effect may take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to initiation of therapy according to the present methods. For example, the beneficial effect may take the form of slowing, stabilizing, stopping, or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. An effective treatment may, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or stop tumor growth, and/or prevent or delay tumor recurrence or recurrence.

The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or combination of agents that provides a desired biological, therapeutic, and/or preventative result. That result may be a reduction, amelioration, palliation, alleviation, delay, and/or relief of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. With respect to tumor development, an effective amount is an amount sufficient to delay the onset of the tumor. With respect to tumor recurrence, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of a drug or composition may be capable of (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, delaying, slowing, or stopping, to some extent, cancer cell infiltration into peripheral organs; (iv) inhibiting tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the development and/or recurrence of a tumor; and/or (vii) alleviating to some extent one or more symptoms associated with the cancer. In one aspect, an "effective amount" is an amount of an antibody or a functional portion, derivative, and/or analog thereof disclosed herein that affects a reduction in cancer (e.g., a reduction in the number of cancer cells); a slowing of cancer progression; or a prevention of cancer regrowth or recurrence. As previously mentioned herein, the combination of the antibody or functional portion, derivative, and/or analog thereof of the present disclosure that binds to EGFR or binds to EGFR and LGR5 and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor, or SN-38 is referred to herein as a "therapeutic agent." In a particular aspect, the effective amount of the antibody or functional portion, derivative, and/or analog thereof of the present disclosure that binds to EGFR or binds to EGFR and LGR5 is a flat dose of 1500 mg administered biweekly to a subject having a cancer of the present disclosure.

As used herein, the term "flat dose" refers to a dosing regimen in which a subject is administered a fixed amount of a therapeutic agent over multiple administrations, regardless of the subject's body weight. Flat dosing is typically abbreviated as qnw, where n is an integer representing the interval and w is weeks. For example, a q2w flat dose dosing regimen of 1500 mg of antibody means that a fixed amount of 1500 mg of antibody is administered every two weeks. As used herein, the therapeutic agent is an antibody that binds EGFR and LGR5, preferably administered in a 1500 mg q2w dosing regimen. In certain aspects, the subject has been administered at least three q2w flat doses of 1500 mg. In certain aspects, the administration is at least four doses and may continue until the patient exhibits sufficient clinical or radiological progression.

The uniform dose may be pre-medicated, meaning that the subject is administered a medication prior to administration of the antibody of the present disclosure. In certain embodiments, the 1500 mg uniform dose of antibody is pre-medicated with an antihistamine, an analgesic, an antipyretic, and/or an anti-inflammatory agent.

An effective amount of combination therapy refers to a combination of an antibody or functional portion, derivative and/or analog thereof of the present disclosure and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38, administered according to the methods described herein, and the order of administration, the amount administered and the frequency of administration are appropriate to effect the treatment.

As used herein, the terms "synergy," "therapeutic synergy," and "synergistic effect" refer to the phenomenon in which treatment of a patient with a combination of therapeutic agents (e.g., a fluoropyrimidine, a platinum-based chemotherapy, a BCL-2 inhibitor, or an EGFR/LGR5 binding antibody such as petosemtamab in combination with SN-38) results in a therapeutically superior outcome than that achieved by each component of the combination alone (see, e.g., T. H. Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). In this context, a therapeutically superior outcome includes one or more of the following: (a) an increase in therapeutic response that is greater than the individual effects of either or both agents when administered alone at the same dose as in the combination; (b) a reduction in the dose of one or more of the agents in the combination without a decrease in therapeutic efficacy; (c) a therapeutic benefit equal to or greater than that achieved when each agent is administered alone at the same dose as in combination, while reducing the incidence of adverse events; (d) a therapeutic benefit greater than that achieved when each agent is administered alone, while reducing dose-limiting toxicities; (e) a delay or minimization of the induction of drug resistance. In organoid models, therapeutic synergy occurs when the combination, when each component is present at a dose that generally does not exceed its individual maximum tolerated dose, achieves a reduction in organoid growth greater than that achieved when the most effective component is administered alone, when used at its maximum tolerated dose.

All literature and references, including Genbank entries, patents and published patent applications, and websites, described herein are each expressly incorporated by reference into this specification to the same extent as if set forth in whole or in part herein.

For clarity and brevity, features are described herein as being part of the same or separate disclosure, although it will be understood that the scope of the disclosure may include preferred aspects having combinations of all or some of the described features.

The present invention will now be described with reference to the following examples, which are merely illustrative and are not intended to limit the scope of the present disclosure. While the present disclosure has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present disclosure.

List of provisions

1. An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine.

2. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and a fluoropyrimidine.

3. In clause 1 or 2, the fluoropyrimidine is fluorouracil, an antibody or a functional part, derivative and/or analog thereof or a method.

4. In any of the preceding clauses, the antibody or functional part, derivative and/or analogue thereof or method, wherein fluorouracil is administered intravenously in a range of 200 to 3000 mg/m ² .

5. An antibody or functional part, derivative and/or analog thereof or method according to any of the preceding clauses, wherein fluorouracil is administered intravenously as a bolus of 400 mg/m ² on day 1, followed by 2400 mg/m ² to 3000 mg/m ² intravenously as a continuous infusion over 46 hours every two weeks.

6. An antibody or functional part, derivative and/or analog thereof or method, wherein in any of the preceding clauses, fluorouracil is administered by intravenous bolus at 500 mg/m ² on days 1, 8, 15, 22, 29 and 36 during an 8-week cycle.

7. In any of the preceding clauses, the cancer has a mutation in one or more genes selected from APC, KRAS, NRAS, SMAD2, SMAD4, SOX9, PIK3CA or TP53, the antibody or functional part, derivative and/or analog thereof or method.

8. An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine and thymidine phosphorylase inhibitor.

9. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and a fluoropyrimidine and thymidine phosphorylase inhibitor.

10. An antibody or functional part, derivative and/or analogue thereof or method according to clause 8 or 9, wherein the fluoropyrimidine is trifluorothymidine and the thymidine phosphorylase inhibitor is tipiracil.

11. An antibody or a functional part, derivative and/or analogue thereof or a method according to any one of clauses 8 to 10, wherein trifluorothymidine and tipiracil are administered at a molar ratio of 1:0.5 at 35 to 80 mg/m ² twice daily.

12. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 8 to 11, wherein the cancer has a mutation in one or more genes selected from APC, FBXW7, KRAS, NRAS, SMAD2, SMAD3, SMAD4, SOX9, PIK3CA or TP53.

13. An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a platinum-based chemotherapeutic agent.

14. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and/or analog thereof, and a platinum-based chemotherapeutic agent.

15. In clause 13 or 14, the platinum-based chemotherapeutic agent is oxaliplatin, an antibody or a functional part, derivative and/or analog thereof or a method.

16. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 13 to 15, wherein oxaliplatin is administered in a range of 65 to 130 mg/m ² .

17. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 13 to 16, wherein oxaliplatin is administered at 85 mg/m ² every two weeks.

18. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 13 to 17, wherein the cancer has a mutation in one or more genes selected from APC, FBXW7, KRAS, NRAS, SMAD2, SMAD4, SOX9, PIK3CA or TP53.

19. An antibody or a functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a BCL-2 inhibitor.

20. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and/or analog thereof, and a BCL-2 inhibitor.

21. In clause 19 or 20, the BCL-2 inhibitor is venetoclax, an antibody or a functional part, derivative and/or analog thereof or a method.

22. An antibody or functional part, derivative and/or analogue thereof or method according to any one of clauses 19 to 21, wherein venetoclax is administered in a range of 20 to 1200 mg/m ² per day.

23. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 19 to 22, wherein the cancer has a mutation in one or more genes selected from APC, KRAS, NRAS, SMAD2, SMAD4, SOX9, PIK3CA or TP53.

24. An antibody or a functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering SN-38.

25. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and SN-38.

26. In clause 24 or 25, the cancer has a mutation in one or more genes selected from APC, FBXW7, KRAS, NRAS, PIK3CA, SMAD2, SMAD3, SMAD4, SOX9, TCF7L2 and TP53, the antibody or functional part, derivative and/or analog thereof or the method.

27. An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering oxaliplatin, folinic acid and fluorouracil.

28. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and oxaliplatin, folinic acid and fluorouracil.

29. An antibody or functional part, derivative and/or analogue thereof or method according to clause 27 or 28, wherein oxaliplatin is administered at 50 to 200 mg/m ² together with intravenous 200 to 600 mg/m ² of folinic acid, followed by intravenous 1200 to 3600 mg/m ² of fluorouracil.

30. An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering irinotecan, folinic acid and fluorouracil.

31. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR or a functional portion, derivative and/or analog thereof and irinotecan, folinic acid and fluorouracil.

32. The antibody or functional part, derivative and/or analogue thereof or method according to clause 30 or 31, wherein irinotecan is administered at 180 mg/m ² together with intravenous 200 to 400 mg/m ² of folinic acid, followed by intravenous 400 to 2400 mg/m ² of fluorouracil.

33. An antibody or functional part, derivative and/or analog thereof or method according to any one of clauses 27 to 32, wherein the cancer has a mutation in one or more genes selected from APC, KRAS, NRAS, SMAD2, SMAD4 or SOX9.

34. In any of the preceding clauses, the cancer is adenocarcinoma or squamous cell carcinoma, the antibody or functional part, derivative and/or analog thereof or the method.

35. In any of the preceding clauses, the cancer is head and neck cancer, stomach, esophagus, gastroesophageal junction cancer, non-small cell lung cancer or colorectal cancer, the antibody or functional part, derivative and/or analog thereof or the method.

36. In any of the preceding clauses, the cancer is colorectal cancer, the antibody or functional part, derivative and/or analog thereof or method.

37. In any of the preceding clauses, the antibody or functional portion, derivative and/or analog thereof or method has enhanced ADCC.

38. In any of the preceding clauses, the antibody or functional portion, derivative and/or analog thereof or method is afucosylated.

39. In any of the preceding clauses, the antibody is a multispecific antibody, an antibody or a functional part, derivative and/or analog thereof, or a method.

40. In any of the preceding clauses, the antibody is a bispecific antibody, an antibody or a functional part, derivative and/or analog thereof, or a method.

41. In any of the preceding clauses, the variable domain that binds to the extracellular portion of EGFR is an antibody or functional part, derivative and/or analog thereof or method, wherein the variable domain is a heavy chain variable region comprising:

- a CDR3 sequence of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3 or a CDR3 sequence which differs by at most 3, 2 or 1 amino acid from the CDR3 sequence of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3; or

- CDR1, CDR2 and CDR3 sequences of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3; or CDR1, CDR2 and CDR3 sequences of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3 having at most 3, 2 or 1 amino acid substitutions; or

- The amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3; or

- The amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3, having at most 15 or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or combinations thereof for the VH chain of MF3370; MF3755; MF4280 or MF4289.

42. An antibody or functional part, derivative and/or analog thereof or method according to any of the preceding clauses, wherein the antibody comprises a second variable domain that does not bind to EGFR.

43. An antibody or functional part, derivative and/or analog thereof or method according to any of the preceding clauses, wherein the antibody comprises a second variable domain that binds LGR5.

44. An antibody or functional part, derivative and/or analog thereof or method, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence depicted in FIG. 1.

45. In any of the preceding clauses, the variable domain that binds to LGR5 is an antibody or functional part, derivative and/or analog thereof or method, wherein the variable domain is a heavy chain variable region comprising:

- a CDR3 sequence of the VH of at least MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3 or a CDR3 sequence which differs by at most 3, 2 or 1 amino acid from the CDR3 sequence of the VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or

- CDR1, CDR2 and CDR3 sequences of VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or CDR1, CDR2 and CDR3 sequences of VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3 having at most 3, 2 or 1 amino acid substitutions; or

- The sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or

- An amino acid sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, as shown in FIG. 3, having at most 15 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or a combination thereof.

46. An antibody or functional part, derivative and/or analog thereof or use or method according to any of the preceding clauses, wherein the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of a variable region selected from the group consisting of MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3, and wherein the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of a variable region selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3.

47. An antibody or functional part, derivative and/or analog thereof or use or method according to any of the preceding clauses, wherein the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of the variable region of MF3755 as depicted in FIG. 3, and wherein the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences of the variable region of MF5816 as depicted in FIG. 3.

48. In any of the preceding clauses, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in Figure 3; or the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in Figure 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5790; MF5803; MF5805 as depicted in Figure 3; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818; or a VH chain MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; as shown in FIG. 3, having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof, relative to said VH. Or an antibody or functional part, derivative and/or analog thereof or use or method comprising the amino acid sequence of MF5818, but preferably wherein the amino acid insertions, deletions and substitutions are not present in the CDR1, CDR2 and CDR3 light chain variable regions.

49. In any of the preceding clauses, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in FIG. 3; or the amino acid sequence of VH chain MF3755 as depicted in FIG. 3 having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof, relative to said VH; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in FIG. 3; An antibody or functional part, derivative and/or analog thereof or use or method comprising the amino acid sequence of VH chain MF5816 as depicted in Figure 3, having at most 15, preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably not more than 5, 4, 3, 2 or 1 amino acid modifications, such as insertions, deletions, substitutions or combinations thereof, preferably wherein the amino acid insertions, deletions and substitutions are not present in the CDR1, CDR2 and CDR3 light chain variable regions.

50. An antibody or functional part, derivative and/or analog thereof or use or method, wherein the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3370; MF3755; MF4280 or MF4289 as depicted in FIG. 3; and wherein the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in FIG. 3.

51. An antibody or functional part, derivative and/or analog thereof or use or method, wherein the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 as depicted in FIG. 3; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5816 as depicted in FIG. 3.

52. An antibody or functional part, derivative and/or analog thereof or use or method according to any of the preceding clauses, wherein both the variable domain that binds EGFR and the variable domain that binds LGR5 comprise CDR1, CDR2 and CDR3 regions of a light chain variable region as illustrated in FIG. 4B.

53. An antibody or functional part, derivative and/or analog thereof or use or method, wherein both the variable domain that binds EGFR and the variable domain that binds LGR5 comprise a light chain variable region as illustrated in FIG. 4B, wherein the variable light chain region comprises 0 to 10 amino acid insertions, deletions, substitutions, additions or combinations thereof, provided that the amino acid insertions, deletions and substitutions are not present in the CDR1, CDR2 and CDR3 light chain variable regions.

54. In any of the preceding clauses, the antibody is petosemtamab, the antibody or a functional part, derivative and/or analog thereof or the method.

55. An antibody or functional part, derivative and/or analog thereof or method, wherein the antibody is a monovalent antibody that does not comprise a second variable domain, or wherein the antibody comprises the EGFR binding variable domain as the only variable domain.

56. In any of the preceding clauses, the subject is a mammal, in particular a human, an antibody or a functional part, derivative and/or analog thereof or a method.

57. In any of the preceding clauses, the treatment comprises providing to the subject 1500 mg of the antibody or functional part, derivative and/or analog thereof or the method.

58. In any of the preceding clauses, the subject to be treated has not received any previous anticancer therapy, the antibody or functional part, derivative and/or analog thereof or method.

59. In any of clauses 1-57, the subject to be treated has progressed after previous anticancer therapy, the antibody or functional part, derivative and/or analog thereof or method.

60. In any of the preceding clauses, the previous anticancer therapy is chemotherapy, targeted anticancer therapy, immunotherapy or radiotherapy, an antibody or a functional part, derivative and/or analog thereof or a method.

61. In any of the preceding clauses, the previous anticancer therapy is fluorouracil, a platinum-based chemotherapy agent, oxaliplatin, FOLFOX, FOLFIRI, TAS-102, trastuzumab, pembrolizumab, nivolumab, cetuximab or a combination thereof, an antibody or a functional part, derivative and/or analog thereof or a method.

62. In any of the preceding clauses, the subject has progressed after fluoropyrimidine-oxaliplatin-based chemotherapy with or without bevacizumab, the antibody or functional part, derivative and/or analog thereof or the method.

63. In clause 62, the subject is an antibody or a functional part, derivative and/or analog thereof or a method to which FOLFIRI is administered.

64. In any of the preceding clauses, the subject has progressed after fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab, the antibody or functional part, derivative and/or analog thereof or the method.

65. In clause 64, the subject is an antibody or a functional part, derivative and/or analog thereof or a method to which FOLFOX is administered.

66. In any of the preceding clauses, the cancer is metastatic colorectal cancer (mCRC), e.g., adenocarcinoma of the colon or rectum, an antibody or functional part, derivative and/or analog thereof, or a method.

67. In any of the preceding clauses, the antibody or functional part, derivative and/or analog thereof and the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38 are administered to the subject simultaneously, separately or sequentially, or the antibody or functional part, derivative and/or analog thereof or the method.

68. A method of treating cancer in a subject of any of the preceding clauses, comprising administering to the subject an effective amount of the antibody or a functional part, derivative and/or analog thereof and an effective amount of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

69. A pharmaceutical composition comprising an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding clauses and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

70. A pharmaceutical composition according to clause 69, wherein the antibody or a functional part, derivative and/or analog thereof is provided as a single formulation comprising a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

71. A pharmaceutical composition according to clause 69, wherein the antibody or a functional part, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 are provided in separate formulations.

72. As a kit part,

- an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding provisions;

- Fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38; and

- A kit part comprising instructions for use of the antibody or a functional part, derivative and/or analog thereof and instructions for use of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

73. In clause 72, the instructions for use of the antibody or its functional part, derivative and/or analogue are a kit part comprising instructions for administration of 1500 mg.

74. A kit part according to clause 72 or 73, comprising instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of adenocarcinoma or squamous cell carcinoma.

75. A kit comprising instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of head and neck cancer, stomach cancer, esophageal cancer, gastroesophageal junction cancer, non-small cell lung cancer or colorectal cancer, according to any one of clauses 72-74.

76. A kit comprising instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of colorectal cancer, according to any one of clauses 72-75.

77. A combination of an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding clauses with a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 for use in the treatment of cancer in a subject in need thereof.

78.

- an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding provisions;

- Instructions for use in the treatment of cancer in the subject of the above antibody and

- A combination of a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 as defined in any of the preceding clauses in the treatment of cancer in a subject.

79. In clause 78, the instructions for use include the amount of the antibody or functional part thereof, derivative and/or analogue to be used and/or the amount and/or administration interval of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and/or the combination including the cancer to be treated.

80.

- Fluoropyrimidines, platinum-based chemotherapeutics, BCL-2 inhibitors or SN-38 as defined in any of the preceding clauses;

- Instructions for use in the treatment of cancer in subjects receiving fluoropyrimidines, platinum-based chemotherapeutics, BCL-2 inhibitors or SN-38;

- Instructions for use in the treatment of cancer in a subject of an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding clauses.

81. In clause 80, the instructions for use include the amount of the antibody or functional part thereof, derivative and/or analogue to be used and/or the amount and/or administration interval of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and/or the combination including the cancer to be treated.

82. Use of an antibody or a functional part, derivative and/or analog thereof as defined in any of the preceding clauses and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 as defined in any of the preceding clauses in the manufacture of one or more medicaments for the treatment of cancer in a subject.

83. In clause 82, the treatment comprises administering to the subject an effective amount of an antibody or a functional part, derivative and/or analog thereof and an effective amount of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38.

84. Use according to clause 82 or 83, wherein the treatment comprises administering to the subject simultaneously, separately or sequentially an antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38.

Example

As used herein, "MFXXXX" (wherein X is independently a number 0 to 9) refers to a Fab comprising a variable domain, wherein the VH has an amino acid sequence identified by the four digits depicted in FIG. 3. Unless otherwise indicated, the light chain variable region of the variable domain typically has the sequence of FIG. 4B. The light chain of an embodiment has the sequence as depicted in FIG. 4A. "MFXXXX VH" refers to an amino acid sequence of the VH identified by the four digits. The MF further comprises a constant region of the light chain and a constant region of the heavy chain that typically interacts with the constant region of the light chain. The variable regions of the VH/heavy chains are different, and typically the CH3 regions are also different, wherein one of the heavy chains has a KK mutation in its CH3 domain and the other has a complementary DE mutation in its CH3 domain (see, for example, PCT/NL2013/050294 (published as WO2013/157954) and FIGS. 5d and 5e ). The bispecific antibodies of the embodiments have a KK/DE CH3 heterodimerization domain as shown in FIG. 5 , an Fc tail having a CH2 domain and a CH1 domain, a common light chain as shown in FIG. 4a , and a VH as indicated by the MF numbers. For example, the bispecific antibody designated MF3755 x MF5816 has a variable domain having the general sequence and a VH having the sequence of MF3755 and a VH having the sequence of MF5816.

The amino acid and nucleic acid sequences of various heavy chain variable regions (VH) are shown in Figure 3. Among other LGR5 and EGFR combinations as depicted in Figure 3, a bispecific antibody EGFR/LGR5, MF3755xMF5816, comprising heavy chain variable regions MF3755 and MF5816 and a common light chain and modifications for enhanced ADCC from afucosylation, was shown to be effective in WO2017/069628.

Example 1: Generation of bispecific antibodies

To ensure efficient heterodimerization and formation of bispecific antibodies, the bispecific antibodies were generated via transient co-transfection of two plasmids encoding IgGs with different VH domains using proprietary CH3 engineering technology. The common light chain was also co-transfected into the same cell, either on the same plasmid or on another plasmid. In the present applications (e.g., WO2013/157954 and WO2013/157953; incorporated herein by reference), the inventors disclosed methods and means for producing bispecific antibodies from a single cell, thereby providing means that favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously employed in the present disclosure. Specifically, desirable mutations for producing essentially only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 of the first CH3 domain (the 'KK-variant' heavy chain), for example L351K and T366K (numbering according to EU numbering) and amino acid substitutions at positions 351 and 368 of the second CH3 domain (the 'DE-variant' heavy chain), for example L351D and L368E, or vice versa (see Figures 5d and 5e). In the cited application, it was previously demonstrated that the negatively charged DE-variant heavy chain and the positively charged KK-variant heavy chain preferentially pair to form heterodimers (so-called 'DEKK' bispecific molecules). Homodimerization of DE-mutant heavy chains (DE-DE homodimers) or KK-mutant heavy chains (KK-KK homodimers) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

The VH gene of the variable domain that binds LGR5 described above was cloned into a vector encoding a positively charged CH3 domain. The VH gene of the variable domain that binds EGFR, as disclosed in WO 2015/130172 (incorporated herein by reference), was cloned into a vector encoding a negatively charged CH3 domain. Suspension-adapted 293F Freestyle cells were cultured in T125 flasks on a shaker at a density of 3.0 × 10e6 cells/ml until plateau. Cells were seeded into each well of a 24-deep well plate at a density of 0.3 to 0.5 × 10e6 viable cells/ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies cloned into a proprietary vector system. Seven days after transfection, the cell supernatant was harvested and filtered through a 0.22 μM filter (Sartorius). Sterile supernatant was used for 4 min until the antibody was purified. It was kept in .

IgG purification and quantification

Purification was performed under sterile conditions in a filter plate using protein A affinity chromatography. First, the pH of the medium was adjusted to pH 8.0, and the IgG-containing supernatant was incubated with protein A Sepharose CL-4B beads (50% v/v) (Pierce) on a shaking platform at 600 rpm at 25°C for 2 hours. The beads were then collected by filtration. The beads were washed twice with PBS, pH 7.4. The bound IgG was then eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris, pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 Multiplate (Millipore). The samples were finally collected in PBS, pH 7.4. IgG concentrations were measured using an Octet. The protein samples were stored at 4°C.

To determine the amount of purified IgG, antibody concentration was determined by Octet analysis using a protein-A biosensor (Forte-Bio, according to the supplier's recommendations) using total human IgG (Sigma Aldrich, catalog no. I4506) as a standard.

The following bispecific antibodies are suitable for use in the present examples and for use in the methods of the present invention: MF3370xMF5790, MF3370xMF5803, MF3370xMF5805, MF3370xMF5808, MF3370xMF5809, MF3370xMF5814, MF3370xMF5816, MF3370xMF5817, MF3370xMF5818, MF3755xMF5790, MF3755xMF5803, MF3755xMF5805, MF3755xMF5808, MF3755xMF5809, MF3755xMF5814, MF3755xMF5816, MF3755xMF5817, MF3755xMF5818, MF4280xMF5790, MF4280xMF5803, MF4280xMF5805, MF4280xMF5808, MF4280xMF5809, MF4280xMF5814, MF4280xMF5816, MF4280xMF5817, MF4280xMF5818, MF4289xMF5790, MF4289xMF5803, MF4289xMF5805, MF4289xMF5808, MF4289xMF5809, MF4289xMF5814, MF4289xMF5816, MF4289xMF5817 and MF4289xMF5818. Each bispecific antibody comprises two VHs as indicated by MF numbers capable of binding to EGFR and LGR5, respectively, and further comprises an Fc tail having a KK/DE CH3 heterodimerization domain represented by SEQ ID NO: 117 ( FIG. 5D ) and SEQ ID NO: 118 ( FIG. 5E ), a CH2 domain represented by SEQ ID NO: 116 ( FIG. 5C ), a hinge domain represented by SEQ ID NO: 115 ( FIG. 5B ) and a CH1 domain represented by SEQ ID NO: 114 ( FIG. 5A ), and a common light chain represented by SEQ ID NO: 107 ( FIG. 4A ).

Example 2: Materials and Methods

Organoid models:

Tumor organoid models were obtained from HUB Organoids (see huborganoids.nl) as described previously [van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients, Cell 161, 933–945 (2015)]. Mutations in components of four major CRC regulatory pathways (WNT, RTK/RAS, TP53, and transforming growth factor-β) in each model are summarized in Table 4. Detailed clinicopathological features can be found in [Herpers B. et al. Nat Cancer. 2022 Apr;3(4):418–436].

Culture medium: Organoids were expanded and exposed by dropping Matrigel Matrix Basement Membrane (Corning, catalog no. 354230) into Organoid Culture Medium (OCM), and then seeded into 384-well plates (Greiner, catalog no. 781091) using a liquid handler. Briefly, OCM was composed of Advanced DMEM (Thermo Fisher Scientific, catalog no. 12634028) containing Pen/Strep (Thermo Fisher Scientific, catalog no. 15140122), Hepes (Thermo Fisher Scientific, catalog no. 15630-056), and Glutamax (Thermo Fisher Scientific, catalog no. 35050038), supplemented with RSPO3 (BioTechne, 3500-RS/CF), Noggin (Peprotech, 120-10C), B27 (Thermo Fisher Scientific, catalog no. 17504001), nicotinamide (Sigma Aldrich, catalog no. N0636), N-acetylcysteine (Sigma Aldrich, catalog no. A9165), SB-202190 (Sigma Aldrich, catalog no. S7067), Gastrin (Merck, catalog no. G9145), and A83-01 (Tocris, catalog no. 2939) was included. For a healthy colon organoid model, OCM was supplemented with WNT3A-conditioned medium (OL-016_v2). 10 μM Y27632 (RhoKi) (Bioconnect, catalog no. M1817) was used during subculture.

Colon organoids were seeded in 384-well plates as 10- to 20-cell stage cell clusters of 300 or 600 cells (model P18T) onto hydrogel. After 30 minutes of gelation, medium was added. For colon tumor types, organoid culture medium was used. For normal colon organoids, organoid culture medium supplemented with WNT3A was used. Growth factors were added to the culture medium at the indicated concentrations on the day of seeding. Bispecific antibodies were added to the growth factor-containing medium on the day of seeding. Compound exposure was performed on the day of seeding or 24 hours after seeding. Organoids were exposed to growth factors, antibodies, and compounds for 5 to 8 days (depending on the growth rate of the organoid model—normal organoids exhaust culture medium more quickly than colon tumor types). After the experiment, organoids were fixed and stained for the actin cytoskeleton and nuclei. Image-based analysis was performed by capturing 25–40 sections per well at a step size of 50 μm per z-stack using Ominer® with a 4x objective (1080 × 1080 pixels). Data per well were aggregated, and the mean and standard deviation per morphological feature (>500 features) were recorded and processed.

Growth factor: EGF (Peprotech. Catalog No. AF-100-15) was used at 10 ng/ml during tumor model expansion and 50 ng/ml for healthy organoid models.

Test compound:

Petosemtamab: 22.9 mg/ml (Lot: FB01701; Store at 4°C); PG2708p218: 2.5 mg/ml (Store at 4°C); 5-FU (F-2449): 10 mM (DMSO) (Lot: G1264; Store at -20°C); Oxaliplatin (15604985): 12.59 mM (PBS) (Lot: 211E0081; Store at 4°C); SN-38 (S4908): 10 mM (DMSO) (Lot: 86639-52-3-02; Store at -20°C); TAS-102 (S8539): 10 mM (DMSO) (Lot: S853901; Store at -20°C); Venetoclax (S8048): 10 mM (DMSO) (Lot: S804806; store at 20°C); Sutaurosporine (HY-15141): 10 mM (DMSO) (Lot: CS2716; store at -20°C).

Video analysis

After incubation, organoids were fixed and stained to visualize nuclei and the actin cytoskeleton, respectively. After washing with PBS, the plates were imaged at 4x magnification using a Molecular Devices ImageXpress Micro XLS system, with 25 to 40 sections per well at a z-step size of 50 μm. Images were processed to quantify the shape, size, location, number, and intensity of each nucleus, organoid, lumen, and epithelium within the organoids.

Data quality control (QC) was performed by bootstrapping the segmented images and the data from the positive and negative controls, using a randomized Z' factor and one-way ANOVA as a measure of data consistency. Aggregated data were normalized (z-scores and control ratios (POC) using DMSO + PBS + PG2708 as negative controls) and processed in TIBCO Spotfire® or MS Excel, formatting the data for graphical visualization and curve fitting in GraphPad Prism v8.4.

Example 3: Dose range testing of individual compounds

To prepare for the combination study in Experiment 4, a range of doses of petosemtamab, 5-FU, oxaliplatin, SN-38, TAS-102, and venetoclax were tested in the presence of 5 ng/ml EGF to estimate the IC20. The models tested were P18T, C55T, C82T, and C82N. PBS, DMSO, and the PG2708 negative control antibody were used as negative controls. 1 μM staurosporine and 0 ng/ml EGF were used as positive controls. For the C82N, C82T, C55T, and P18T organoid models, exposure to compounds was performed on the day of culture, with an exposure time of 8 days. Table 5 shows the various concentrations tested for each compound.

The control group behaved as expected. All compounds decreased organoid proliferation, increased cell death, and reduced lumen formation as the compound dose increased. Based on the collected results, the IC20 values of all compounds were determined during the manufacturing process of Example 4. The IC20 values are summarized in Table 6.

Example 4: Combination study of petosemtamab and individual compounds

Whether petosemtamab treatment sensitized organoids to 5-FU, oxaliplatin, SN-38, TAS-102, or venetoclax treatment in a 6x6 complex matrix at 5 ng/ml EGF concentration was tested in models P18T, C20T, C55T, C82T, C92T, and C92N.

The following combinations were added on the day of seeding: fetosemtamab (0 - 0.031 - 0.1 - 0.316 - 1 - 3.162 μg/ml); 5-FU, TAS-102, or venetoclax at 0 - 0.1 - 0.316 - 1 - 3.162 - 10 μM; or SN38 at 0 - 0.1 - 0.316 - 1 - 3.162 - 10 nM; or oxaliplatin at 0 - 0.316 - 1 - 3.162 - 10 - 31.62 μM.

Antibody-drug combinations were incubated for 7 days prior to fixation to visualize 3D actin and nuclear organization in organoids. PBS, DMSO, and the PG2708 negative control antibody were used as negative controls. 1 μM staurosporine and 0 ng/ml EGF were used as positive controls.

For each model and combination, three characteristics were evaluated: nuclei count, sum of progeny nuclei (total number of viable nuclei), and total organoid size. Total organoid size is known to decrease upon growth inhibition and induction of apoptosis.

The control group behaved as expected and remained consistent throughout the experiment. All compound combinations reduced organoid growth and increased cell death. Total organoid size was additionally used to assess the synergistic effects between the various combinations.

Data Analysis :

Data were analyzed using the online tool SynergyFinder.org (Zheng, S. et. al. bioRxiv 2021.06.01.446564 (2021); Zheng S, et. al. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics Proteomics Bioinformatics. 2022 Jan 25:S1672-0229(22)00008-0). The SynergyFinder tool uses four methods to indicate the degree of interaction: HSA, LOEWE, BLISS, and ZIP. After SynergyFinder analysis, the CI of each data point was calculated using the Chou-Talalay method (refer to the literature [Chou T.C. Drug combination using the Chou-Talalay method. Cancer research 70, 440-446 (2010)]). The CI was calculated for each data point by dividing the maximum of the measured single-agent effect or the model-predicted single-agent effect by the actual effect measured using all four methods (ZIP, BLISS, LOEWE, and HSA). The synergy scores and CI scores for each model are summarized in Table 7.

In summary, combination therapy with petosemtamab and the five standard-of-care drugs tested induced synergistic growth inhibition in the indicated models.

Example 5: Combination study of petosemtamab and Folfox/Folfiri

Additionally, we tested whether combination therapy with petosemtamab and FOLFOX and FOLFIR enhanced tumor regression efficacy. The individual components of the FOLFOX and FOLFIRI combination drugs are described elsewhere herein. In patients, the first treatment was oxaliplatin 85 mg/m ² intravenously administered over 2 hours. This correlates with a plasma Cmax of 1.61 to 2.2 μg/ml, or 4 to 5 μM. Next, leucovorin was administered intravenously at 180 mg/ ^{m 2} over 90 to 120 minutes in combination with irinotecan. 150 mg/m ² irinotecan maintained the plasma Cmax of irinotecan at approximately 2 μg/ml, and irinotecan provides a mean SN-38 metabolite of 36 ng/ml (92 nM). 5-FU was then administered as a bolus injection of 400 mg/m ² to maintain a mean Cmax of 55 μg/ml, followed by an IV infusion of 2400 mg/m ² over 46 hours. The literature indicates that a 3-day IV infusion of 1750 mg/m ² results in a mean plasma concentration of 4.6 μM (peak concentration 7.3 μM). Leucovorin or folinic acid was used at a concentration that allowed dissolution (50 μg/ml). This resulted in the following combinations:

- 5 μM 5-FU + 5 μM oxaliplatin + 50 μg/ml folinic acid to mimic FOLFOX (5 μM) or

- 5 μM 5-FU + 100 nM SN-38 + 50 μg/ml folinic acid to mimic FOLFIRI (5 μM).

- The combination was tested in model C55T under conditions containing 5ng/ml EGF.

- The synergistic effect of the combination of FOLFIRI and FOLFOX was calculated using the whole organoid size and the method described in Example 4. The synergistic score and combination index (CI) are summarized in Table 8. The synergistic score and CI indicate that both FOLFIRI and FOLFOX exhibited a synergistic effect when combined with petosemtamab in the indicated model.

Example 6: Clinical Study Protocol

Study design

This is a first-in-human, open-label, multicenter Phase 1/2 study combining an initial dose-escalation and dose-expansion phase in a combination cohort for mCRC. The initial dose-escalation phase has been completed, and the preliminary RP2D was set at 1,500 mg Q2W.

The dose expansion portion of this study evaluates the combination of petosemtamab and selected chemotherapeutic agents for the primary treatment of mCRC.

Petosemtamab will be administered IV as a flat dose Q2W over an infusion period of 2 to approximately 6 hours in 4-week cycles (28 days).

Patients will be assigned to dose-expansion arms, including one arm using petosemtamab as a single agent and one arm using petosemtamab in combination with chemotherapy. Specifically, the combination of petosemtamab and FOLFOX will be tested in one arm, and in another arm, in combination with FOLFIRI.

We will analyze safety, pharmacokinetics, immunogenicity, and antitumor activity in all cohort patients, and perform retrospective biomarker analysis including EGFR and LGR5 status.

research group

Inclusion criteria

1. You must sign an informed consent form (ICF) before starting any research procedure.

2. Age 18 or older at the time of signing the ICF.

3. Histologically or cytologically confirmed solid tumor with evidence of metastatic or locally advanced disease that is not amenable to standard treatment with curative intent.

Expansion cohort: Patients with locally advanced, unresectable or metastatic disease for the following indications:

o Patients with mCRC receiving second-line therapy must have previously been diagnosed with unresectable or metastatic colon or rectal adenocarcinoma, either histologically or cytologically confirmed. Patients must be wild-type for RAS/RAF as determined using next-generation sequencing (NGS) on tumor tissue (primary or metastatic). NGS data reports will be provided for sponsor verification prior to enrollment. Patients must be naïve to prior anti-EGFR therapy. Radiographically confirmed disease progression must have occurred during or within 6 months of the previous 1L chemotherapy.

o Cohort treated with petosemtamab and FOLFIRI: Patients must have received only one prior chemotherapy regimen consisting of 1L fluoropyrimidine-oxaliplatin-based chemotherapy with or without bevacizumab for metastatic disease.

o Cohort treated with petosemtamab and FOLFOX: Patients must have received only one prior chemotherapy regimen consisting of 1L fluoropyrimidine-irinotecan-based chemotherapy with or without bevacizumab for metastatic disease.

4. A fresh tumor sample (formula-fixed, paraffin-embedded [FFPE]) at baseline from a metastatic or primary site. If the patient has available tumor samples as FFPE blocks with sufficient material (at least 20 slides with >20% tumor content) and has not received any additional chemotherapy since sample collection, a fresh tumor biopsy is not required at baseline. Archival FFPE slides are not acceptable.

5. Measurable disease as defined by RECIST v1.1 by radiological methods.

6. ECOG PS 0 or 1.

7. Life expectancy of 12 weeks or more.

8. Left ventricular ejection fraction (LVEF) greater than 50% as determined by echocardiography (ECHO) or multi-gated acquisition (MUGA) scan.

9. Proper organ function:

Absolute neutrophil count (ANC) ≥1.5×10 ⁹ /L

Hemoglobin ≥9 g/dL

Platelets ≥100×10 ⁹ /L

Serum magnesium, sodium, corrected total calcium, phosphate, and potassium within normal ranges (or corrected with supplements or appropriate treatment). If electrolyte levels remain outside the normal range despite corrective treatment, the client should be contacted to determine eligibility.

Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤2.5×the upper limit of normal (ULN) and total bilirubin ≤1.5×ULN (except due to known Gilbert syndrome, in which case total bilirubin >3.0×ULN or direct bilirubin >1.5×ULN would be excluded); in cases of liver involvement, ALT/AST ≤5×ULN and total bilirubin ≤2×ULN would be acceptable, except in cases in which total bilirubin ≤3.0×ULN or direct bilirubin ≤1.5×ULN would be acceptable due to known Gilbert syndrome.

Serum creatinine ≤1.5×ULN or creatinine clearance (CrCl) ≥60 mL/min (calculated according to the Cockroft and Gault formula or the Modified Diet for Renal Disease (MDRD) formula for patients older than 65 years).

International normalized ratio (INR) or prothrombin time (PT) ≤1.5×ULN, unless the patient is receiving anticoagulant therapy and is within the therapeutic range of the anticoagulant used intentionally.

Activated partial thromboplastin time (APTT) or partial thromboplastin time (PTT) ≤1.5×ULN, unless the patient is receiving anticoagulant therapy and is within the therapeutic range of the anticoagulant used intentionally.

Exclusion criteria

1. Central nervous system metastases that are untreated, symptomatic, or require radiation therapy, surgery, or continuous steroid therapy within 14 days of study participation.

2. Known meningeal invasion.

3. Participation in additional clinical trials or treatment with any investigational drug within 4 weeks prior to study participation.

4. Any systemic anticancer treatment within 4 weeks or 5 half-lives (whichever is longer) of the first dose of the study treatment. For cytotoxic agents with major delayed toxicities (e.g., mitomycin C, nitrosoureas) or anticancer immunotherapy, a 6-week washout period is required.

5. Requirements for immunosuppressive medications (e.g., methotrexate, cyclophosphamide).

6. Major surgery or radiation therapy within 3 weeks of the first dose of study treatment. Patients who have previously received radiation therapy to more than 25% of the bone marrow are ineligible, regardless of the timing of administration.

7. Clinically significant toxicity >1 of sustained grade (except alopecia) related to previous antineoplastic therapy; Stable sensory neuropathy ≤2 according to National Cancer Institute - Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03/v5.0 or criteria valid at the time of administration are acceptable.

8. History of hypersensitivity to any of the excipients of petosemtamab, human proteins, or non-IMP treatments required for this study.

9. Uncontrolled hypertension (systolic blood pressure [BP] >150 mmHg and/or diastolic blood pressure >100 mmHg) despite adequate treatment; unstable angina; congestive heart failure of New York Heart Association (NYHA) class II to IV or history of serious cardiac arrhythmia requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia); or history of myocardial infarction within 6 months prior to study entry.

10. History of previous malignancy, excluding resected cervical intraepithelial neoplasia or nonmelanoma skin cancer, or a history of cured cancer or second primary malignancy with no evidence of disease for more than 3 years and judged to be at low risk of recurrence.

11. Other conditions requiring continuous oxygen therapy, including patients with dyspnea at rest regardless of cause, a history of interstitial lung disease (ILD) (e.g., pneumonia or pulmonary fibrosis), or evidence of ILD on baseline chest computed tomography (CT).

12. Current serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorders.

13. Patients with known infectious diseases

14. Pregnant or lactating patients; Patients of childbearing potential must use highly effective contraception before, during, and for 6 months after the last dose of petosemtamab.

15. Patients with mCRC who have RAS/RAF mutations identified in local ctDNA or tumor examination at screening or in their medical history are not eligible for participation in this study.

16. For the mCRC cohort; patients with active inflammatory bowel disease or other intestinal diseases causing chronic diarrhea (defined as NCI-CTCAE grade 2 or higher) were not eligible for this study.

17. Patients with peripheral sensory neuropathy with functional impairment (defined as NCI CTCAE grade 3 or higher) are not eligible for this study.

Pre-screening of patients

Pre-screening can be performed to exclude somatic mutations in RAS and RAF family genes (e.g., KRAS, NRAS, HRAS, BRAF, ARAF, RAF1 wild type).

Prescreening of mCRC patients eligible for second-line treatment with FOLFIRI or FOLFOX can be used to determine eligibility, with validated tumor NGS assay results used to confirm the absence of RAS/RAF or other relevant mutations in the patient's tumor. This report can be generated at any time during the natural course of the disease. Patients must have known RAS/RAF status to be included in this cohort.

Petosemtamab administration

Petosemtamab is administered Q2W as an IV infusion using a 1,500 mg Q2W dosing regimen. For the Cycle 1 Day 1 infusion, the infusion is administered IV over approximately 6 hours. Subsequent infusions after Cycle 1 Day 1 may be shortened to 2 hours (±15 minutes) at the investigator's discretion and in the absence of adverse reaction rates (IRRs). Alternatively, petosemtamab is used as a flat dose of 1,100 mg Q2W or administered in an amount that achieves at least 90%, at least 95%, or at least 99% human receptor target engagement for both EGFR and LGR5 in a statistically significant number of subjects across relevant body weights. The target binding of 90% can be achieved using a flat dose of about 1000 mg Q2W. The target binding of 95% can be achieved using a flat dose of about 1100 mg to about 1200 mg Q2W.

The cycle is considered to be 4 weeks. For each patient, a 1-hour observation period will be implemented after the end of infusion (EOI).

Treatment period

Study treatment will be administered until progressive disease (PD) confirmed by independent review (as per RECIST v1.1), unacceptable toxicity, patient withdrawal of consent, patient noncompliance, investigator decision (e.g., clinical deterioration), or discontinuation of petosemtamab for more than 6 consecutive weeks. All patients entered a survival follow-up phase to be monitored for survival until the end of the study, and all patients were eligible for at least 18 months of follow-up after initiation of new anticancer treatment.

Efficacy evaluation

Tumor evaluation will be performed every 8 weeks using contrast-enhanced CT/MRI of the head and neck, chest, abdomen, and appropriate anatomy of the tumor site according to RECIST v1.1 for up to 12 months after the start of treatment. After 12 months, tumor evaluation can be performed using Q12W. If clinically indicated, a brain scan should be performed at baseline and, if metastases are present, repeated at the same frequency as the head and neck, chest, and abdomen scans. For patients with suspected bone metastases at baseline or with suspicious lesions on examination outside the CT scan field, a bone scan will be performed if clinically indicated. All patient images will be processed at a central imaging center for BICR and reviewed by local investigators.

accompanying medications

Allowed

Petosemtamab is broken down into amino acids by lysosomal enzymes in the liver and/or kidneys and reabsorbed as endogenous proteins. Therefore, drug-drug interactions are not expected with drugs metabolized by cytochrome P450 (CYP450), including components of the FOLFIRI and FOLFOX regimens.

Before the first infusion, appropriate recommended premedication regimens must be followed. For subsequent infusions, the investigator is encouraged to administer appropriate premedication regimens based on their medical judgment. If an IRR, hypersensitivity, or allergic reaction occurs, symptomatic treatment should be administered according to standard local clinical practice and dosing guidelines should be followed.

Any medication necessary for the patient's safety and well-being and not expected to interfere with the study drug evaluation may be administered at the investigator's discretion.

During this study period, concurrent radiotherapy for symptom control without evidence of progression may be administered with the sponsor's approval.

Contraindicated medications include concomitant chronic oral corticosteroids (prednisone equivalents ≥10 mg/day), tumor necrosis factor (TNF)-alpha inhibitors, anti-T-cell antibodies, or other immunosuppressive medications. Strong CYP3A4 inducers, strong CYP3A4, or UGT1A1 inhibitors should not be administered with irinotecan unless other therapeutic options are available. No investigational drugs or other anticancer therapies should be administered during the study. Optionally, herbal medicine for cancer treatment may be initiated. Herbal medicine treatment initiated prior to study entry and continued throughout the study period is permitted. Major surgery or radiation therapy is not permitted without the sponsor's consent except in emergency situations. Prior radiation therapy involving more than 25% of the bone marrow.

Attach an electronic file of the sequence list

Claims (54)

An antibody or a functional part, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR for use in the treatment of cancer in a subject, wherein the treatment further comprises administering a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or a functional portion, derivative and/or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. An antibody or a functional part, derivative and/or analog thereof or a method according to claim 1 or 2, wherein the cancer is adenocarcinoma or squamous cell carcinoma. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 3, wherein the cancer is head and neck cancer, stomach, esophageal, gastroesophageal junction cancer, non-small cell lung cancer or colorectal cancer. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 4, wherein the cancer is colorectal cancer. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 5, wherein the fluoropyrimidine is fluorouracil. In claim 6, the antibody or functional part, derivative and/or analogue thereof or method, wherein the fluorouracil is administered intravenously in a range of 200 to 3000 mg/m ² . An antibody or functional part, derivative and/or analog thereof or method according to claim 6 or 7, wherein the fluorouracil is administered intravenously as a bolus of 400 mg/m ² on the first day and then as a continuous infusion of 2400 mg/m ² to 3000 mg/m ² over 46 hours every two weeks. An antibody or functional part, derivative and/or analog thereof or method according to claim 6 or 7, wherein fluorouracil is administered by intravenous bolus at 500 mg/m ² on days 1, 8, 15, 22, 29 and 36 during an 8-week cycle. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 5, wherein the fluoropyrimidine is trifluridine, and the treatment further comprises a step of administering a thymidine phosphorylase inhibitor. In claim 10, the thymidine phosphorylase inhibitor is tipiracil hydrochloride, an antibody or a functional part, derivative and/or analog thereof or a method. An antibody or a functional part, derivative and/or analog thereof or a method according to claim 10 or 11, wherein the trifluridine and tipiracil hydrochloride are administered at a molar ratio of 1:0.5 at 35 to 80 mg/m ² twice a day. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 5, wherein the platinum-based chemotherapeutic agent is oxaliplatin. In claim 13, the antibody or functional part, derivative and/or analog thereof or method, wherein oxaliplatin is administered at 85 mg/m ² every two weeks. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 5, wherein the BCL-2 inhibitor is venetoclax. In claim 15, venetoclax is administered at 20 to 1200 mg per day, an antibody or a functional part, derivative and/or analog thereof or a method. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 5, wherein the treatment comprises administering SN-38. An antibody or a functional part, derivative and/or analog thereof or a method, in claim 6, wherein the treatment further comprises administering oxaliplatin and folinic acid. An antibody or functional part, derivative and/or analogue thereof or method, in claim 18, wherein the oxaliplatin is administered at 50 to 200 mg/m ² together with intravenous 200 to 600 mg/m ² of folinic acid, followed by intravenous 1200 to 3600 mg/m ² of fluorouracil. An antibody or a functional part, derivative and/or analog thereof or a method, in claim 6, wherein the treatment further comprises administering irinotecantin and folinic acid. An antibody or functional part, derivative and/or analogue thereof or method, in claim 20, wherein the irinotecan is administered at 180 mg/m ^{2 together with intravenous 200 to 400 mg/m 2 of} folinic acid, followed by intravenous 400 to 2400 mg/m ² of fluorouracil. An antibody or functional part, derivative and/or analogue or method thereof, wherein the antibody has enhanced ADCC according to any one of claims 1 to 21. An antibody or functional part, derivative and/or analogue or method according to any one of claims 1 to 22, wherein the antibody is afucosylated. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 23, wherein the antibody is a multispecific antibody. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 24, wherein the antibody is a bispecific antibody. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 25, wherein the variable domain binding to the extracellular portion of EGFR is a heavy chain variable region comprising:
- a CDR3 sequence of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3 or a CDR3 sequence which differs by at most 3, 2 or 1 amino acid from the CDR3 sequence of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3; or
- CDR1, CDR2 and CDR3 sequences of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3; or CDR1, CDR2 and CDR3 sequences of VH of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3 having at most 3, 2 or 1 amino acid substitutions; or
- The amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3; or
- The amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in FIG. 3, having at most 15 or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid insertions, deletions, substitutions or combinations thereof for the VH chain of MF3370; MF3755; MF4280 or MF4289. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 26, wherein the antibody comprises a second variable domain that does not bind to EGFR. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 27, wherein the antibody comprises a second variable domain that binds to LGR5. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 28, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence depicted in FIG. 1. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 29, wherein the variable domain that binds to LGR5 is a heavy chain variable region comprising:
- a CDR3 sequence of the VH of at least MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3 or a CDR3 sequence which differs by at most 3, 2 or 1 amino acid from the CDR3 sequence of the VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or
- CDR1, CDR2 and CDR3 sequences of VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or CDR1, CDR2 and CDR3 sequences of VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3 having at most 3, 2 or 1 amino acid substitutions; or
- The sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in FIG. 3; or
- An amino acid sequence of the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, as shown in FIG. 3, having at most 15 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions, deletions, substitutions, or a combination thereof. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 30, wherein the antibody is petosemtamab. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 26, wherein the antibody is a monovalent antibody that does not comprise a second variable domain, or wherein the antibody comprises the EGFR binding variable domain as the only variable domain. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 32, wherein the subject is a mammal, particularly a human. An antibody or functional part, derivative and/or analog thereof or method according to any one of claims 1 to 33, wherein the treatment comprises providing the subject with 1500 mg of the antibody or functional part, derivative and/or analog thereof. An antibody or a functional part, derivative and/or analog thereof or method according to any one of claims 1 to 34, wherein the subject to be treated has not previously received anticancer therapy. An antibody or a functional part, derivative and/or analog thereof or method according to any one of claims 1 to 34, wherein the cancer or subject to be treated has progressed after previous anticancer therapy. In claim 36, the previous anticancer therapy is chemotherapy, targeted therapy, immunotherapy or radiotherapy, an antibody or a functional part, derivative and/or analog thereof or a method. An antibody or functional part, derivative and/or analog thereof or method according to claim 36 or 37, wherein the previous anticancer therapy is fluorouracil, a platinum-based chemotherapy agent, oxaliplatin, FOLFOX, FOLFIRI, TAS-102, trastuzumab, pembrolizumab, nivolumab, cetuximab or a combination thereof. An antibody or a functional part, derivative and/or analog thereof or a method according to any one of claims 1 to 38, wherein the antibody or a functional part, derivative and/or analog thereof and the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38 are administered to the subject simultaneously, separately or sequentially. A method of treating cancer in a subject according to any one of claims 1 to 39, comprising administering to the subject an effective amount of the antibody or a functional part, derivative and/or analog thereof and an effective amount of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38. A pharmaceutical composition comprising an antibody or a functional part, derivative and/or analog thereof as defined in any one of claims 1 to 40 and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38. A pharmaceutical composition according to claim 41, wherein the antibody or a functional part, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 are provided in a single formulation. A pharmaceutical composition according to claim 41, wherein the antibody or a functional part, derivative and/or analog thereof and the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 are provided in separate formulations. - An antibody or a functional part, derivative and/or analog thereof as defined in any one of claims 1 to 43;
- Fluoropyrimidines, platinum-based chemotherapy, BCL-2 inhibitors, or SN-38; and
- Instructions for use of the antibody or its functional part, derivative and/or analogue and instructions for use of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38
A kit of parts, including: A kit of parts, wherein the instructions for use of the antibody or functional part, derivative and/or analogue thereof in claim 44 include instructions for administration of a dose of 1500 mg. A kit of parts comprising instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of adenocarcinoma or squamous cell carcinoma in accordance with claim 44 or 45. A kit of parts according to any one of claims 44 to 46, wherein the kit comprises instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of head and neck cancer, stomach, esophageal, gastroesophageal junction cancer, non-small cell lung cancer or colorectal cancer. A kit of parts according to any one of claims 44 to 47, wherein the kit comprises instructions for use of the antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 in the treatment of colorectal cancer. A combination of an antibody or a functional part, derivative and/or analog thereof as defined in any one of claims 1 to 48 and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 as defined in any one of claims 1 to 48 for use in the treatment of cancer in a subject in need thereof. - An antibody or a functional part, derivative and/or analog thereof as defined in any one of claims 1 to 49;
- Instructions for use in the treatment of cancer in the subject of the above antibody and
- Instructions for use in the treatment of cancer in a subject of a fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 as defined in any one of claims 1 to 49.
A combination of . In claim 50, the instructions for use include the amount of the antibody or functional part, derivative and/or analog thereof to be used and/or the amount and/or dosing interval of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and/or the cancer to be treated. - Fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 as defined in any one of claims 1 to 51;
- Instructions for use in the treatment of cancer in subjects receiving fluoropyrimidines, platinum-based chemotherapeutics, BCL-2 inhibitors or SN-38; and
- Instructions for use in the treatment of cancer in a subject of an antibody or a functional part, derivative and/or analog thereof as defined in any one of claims 1 to 51.
A combination of . In claim 52, the instructions for use include the amount of the antibody or functional part, derivative and/or analog thereof to be used and/or the amount and/or dosing interval of the fluoropyrimidine, platinum-based chemotherapeutic agent, BCL-2 inhibitor or SN-38 and/or the cancer to be treated. Use of an antibody or a functional part, derivative and/or analog thereof and a fluoropyrimidine, a platinum-based chemotherapeutic agent, a BCL-2 inhibitor or SN-38 as defined in any one of claims 1 to 53 in the manufacture of one or more medicaments for the treatment of cancer in a subject, wherein the treatment comprises administering simultaneously, separately or sequentially the antibody or a functional part, derivative and/or analog thereof and the fluoropyrimidine, the platinum-based chemotherapeutic agent, the BCL-2 inhibitor or SN-38.