Patent Application
20240400717
The present invention relates to an antibody. More particularly, the present invention relates to the antibody for cancer therapy.
Lung cancer is the first and colorectal cancer the third most common cause of cancer death worldwide (Johdi and Sukor 2020; Siegel et al. 2021). Despite the incidence of pancreatic cancer is not as high as lung cancer, it is one of the most aggressive cancers with very low five-year relative survival rate (Li et al. 2021; Siegel et al. 2021). Notably, largely increased non-enzymatic chitinase-3 like-protein-1 (CHI3L1) in serum and intratumoral expression correlates with tumor metastasis and poor outcome of lung, colorectal and pancreatic cancer patients (Yeo et al. 2019; Zhao et al. 2020), making it an appealing target for cancer therapy. In addition, level of CHI3L1 in serum or plasma has been proposed as a non-invasive prognostic biomarker (Kzhyshkowska et al. 2016; Wang et al. 2020).
CHI3L1 is a carbohydrate-binding lectin with no chitinase activity. CHI3L1 modulates inflammatory balance between Th1 and Th2 cells, cytokines induced inflammation, inflammatory cells apoptosis, alternative activated macrophage (M2) differentiation, AKT and extracellular signal-regulated kinase (ERK) signaling (Higashiyama et al. 2019; Kim et al. 2018); (Kim et al. 2020; Letuve et al. 2008). Within all pathways, AKT and ERK are the most widely studied signaling cascades involved in CHI3L1-induced inflammatory response (He et al. 2013). For example, activation of AKT and ERK pathways is essential for restraining pro-inflammation and M2 polarization (Traves et al. 2012; Vergadi et al. 2017).
The creation of immunosuppressive tumor microenvironment (TME) is involved in the CHI3L1-induced cancer cell promotion. CHI3L1 can be secreted by various cells, including macrophages, T cells, fibroblasts, epithelial cells and tumor cells (Cohen et al. 2017; Park et al. 2010; Xing et al. 2017).
Therefore, how to treating CHI3L1-induced cancer, the related art really needs to be improved.
The present disclosure provides an antibody or an antigen-binding portion thereof binding to CHI3L1, comprising: a heavy chain variable (VH) region comprising a heavy chain complementarity determining region (CDR-H1), a CDR H2, and a CDR H3 and a light chain variable (VL) region comprising a light chain complementarity determining region 1 (CDR L1), a CDR L2, and a CDR L3, wherein the CDR-H1 comprises SEQ ID NO: 3, the CDR-H2 comprises SEQ ID NO: 4, and the CDR-H3 comprises SEQ ID NO: 5; and the CDR-L1 comprises SEQ ID NO: 6, the CDR-L2 comprises SEQ ID NO: 7, and the CDR-L3 comprises SEQ ID NO: 8, or wherein the CDR-H1 comprises SEQ ID NO: 11, the CDR-H2 comprises SEQ ID NO: 12, and the CDR-H3 comprises SEQ ID NO: 13; and the CDR-L1 comprises SEQ ID NO: 14, the CDR-L2 comprises SEQ ID NO: 15, and the CDR-L3 comprises SEQ ID NO: 16, or wherein the CDR-H1 comprises SEQ ID NO: 19, the CDR-H2 comprises SEQ ID NO: 20, and the CDR-H3 comprises SEQ ID NO: 21; and the CDR-L1 comprises SEQ ID NO: 22, the CDR-L2 comprises SEQ ID NO: 23, and the CDR-L3 comprises SEQ ID NO: 24, or wherein the CDR-H1 comprises SEQ ID NO: 27, the CDR-H2 comprises SEQ ID NO: 28, and the CDR-H3 comprises SEQ ID NO: 29; and the CDR-L1 comprises SEQ ID NO: 30, the CDR-L2 comprises SEQ ID NO: 31, and the CDR-L3 comprises SEQ ID NO: 32, or wherein the CDR-H1 comprises SEQ ID NO: 35, the CDR-H2 comprises SEQ ID NO: 36, and the CDR-H3 comprises SEQ ID NO: 37; and the CDR-L1 comprises SEQ ID NO: 38, the CDR-L2 comprises SEQ ID NO: 39, and the CDR-L3 comprises SEQ ID NO: 40, or wherein the CDR-H1 comprises SEQ ID NO: 43, the CDR-H2 comprises SEQ ID NO: 44, and the CDR-H3 comprises SEQ ID NO: 45; and the CDR-L1 comprises SEQ ID NO: 46, the CDR-L2 comprises SEQ ID NO: 47, and the CDR-L3 comprises SEQ ID NO: 48, or wherein the CDR-H1 comprises SEQ ID NO: 51, the CDR-H2 comprises SEQ ID NO: 52, and the CDR-H3 comprises SEQ ID NO: 53; and the CDR-L1 comprises SEQ ID NO: 54, the CDR-L2 comprises SEQ ID NO: 55, and the CDR-L3 comprises SEQ ID NO: 56, or wherein the CDR-H1 comprises SEQ ID NO: 59, the CDR-H2 comprises SEQ ID NO: 60, and the CDR-H3 comprises SEQ ID NO: 61; and the CDR-L1 comprises SEQ ID NO: 62, the CDR-L2 comprises SEQ ID NO: 63, and the CDR-L3 comprises SEQ ID NO: 64, or wherein the CDR-H1 comprises SEQ ID NO: 67, the CDR-H2 comprises SEQ ID NO: 68, and the CDR-H3 comprises SEQ ID NO: 69; and the CDR-L1 comprises SEQ ID NO: 70, the CDR-L2 comprises SEQ ID NO: 71, and the CDR-L3 comprises SEQ ID NO: 72, or wherein the CDR-H1 comprises SEQ ID NO: 75, the CDR-H2 comprises SEQ ID NO: 76, and the CDR-H3 comprises SEQ ID NO: 77; and the CDR-L1 comprises SEQ ID NO: 78, the CDR-L2 comprises SEQ ID NO: 79, and the CDR-L3 comprises SEQ ID NO: 80, or wherein the CDR-H1 comprises SEQ ID NO: 83, the CDR-H2 comprises SEQ ID NO: 84, and the CDR-H3 comprises SEQ ID NO: 85; and the CDR-L1 comprises SEQ ID NO: 86, the CDR-L2 comprises SEQ ID NO: 87, and the CDR-L3 comprises SEQ ID NO: 88.
In some embodiments, the antibody or the antigen-binding portion thereof binding to CHI3L1 further comprises the VH region comprising SEQ ID NO: 1 or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 1; and the VL region comprising SEQ ID NO: 2, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 2; the VH region comprising SEQ ID NO: 9, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 9; and the VL region comprising SEQ ID NO: 10, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 10; the VH region comprising SEQ ID NO: 17, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 17; and the VL region comprising SEQ ID NO: 18, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 18; the VH region comprising SEQ ID NO: 25, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 25; and the VL region comprising SEQ ID NO: 26, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 26; the VH region comprising SEQ ID NO: 33, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 33; and the VL region comprising SEQ ID NO: 34, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 34; the VH region comprising SEQ ID NO: 41, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 41; and the VL region comprising SEQ ID NO: 42, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 42; the VH region comprising SEQ ID NO: 49, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 49; and the VL region comprising SEQ ID NO: 50, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 50; the VH region comprising SEQ ID NO: 57, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 57; and the VL region comprising SEQ ID NO: 58, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 58; the VH region comprising SEQ ID NO: 65, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 65; and the VL region comprising SEQ ID NO: 66, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 66; the VH region comprising SEQ ID NO: 73, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 73; and the VL region comprising SEQ ID NO: 74, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 74; or the VH region comprising SEQ ID NO: 81, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 81; and the VL region comprising SEQ ID NO: 82, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO: 82.
In some embodiments, the antibody is a human antibody.
In some embodiments, the antibody or the antigen-binding portion thereof is a single chain Fv (scFv), fragment-antigen binding (Fab), (Fab′) 2, or (scFv′) 2.
In some embodiments, the antibody is an IgG, IgE, IgM, IgD, IgA, or IgY antibody.
In some embodiments, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.
The present disclosure also provides a pharmaceutical composition, comprising the antibody or the antigen-binding portion thereof as above mentioned, and at least one pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical composition further comprises a therapeutic agent.
In some embodiments, the therapeutic agent comprises gemcitabine.
The present disclosure also provides an antibody-drug conjugate comprising a therapeutic agent; and an antibody or an antigen-binding portion thereof as above mentioned, wherein the therapeutic agent is covalently conjugated to the antibody or the antigen-binding portion thereof by a linker.
The present disclosure also provides a nucleic acid molecule encoding the antibody or the antigen-binding portion thereof as above mentioned.
The present disclosure also provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of the antibody or the antigen-binding portion thereof as above mentioned.
In some embodiments, the cancer is a CHI3L1 expressing tumor.
In some embodiments, the cancer is pancreas cancer, colon cancer, lung cancer, prostate cancer, non-small cell lung cancer (NSCLC), melanoma, lymphoma, breast cancer, esophageal cancer, head and neck cancer, renal cell carcinoma (RCC), ovarian cancer, kidney cancer, urinary bladder cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, stomach cancer, rectal cancer, oral cavity cancer, pharynx cancer, thyroid cancer, skin cancer, brain cancer, bone cancer, hematopoietic cancer, or leukemia.
In some embodiments, the antibody or the antigen-binding portion thereof is used in combination with a therapeutic agent.
In some embodiments, the therapeutic agent comprises gemcitabine.
In some embodiments, the antibody or the antigen-binding portion thereof inhibits activated AKT signal, inhibits activated ERK signal, inhibits cancer cells migration, inhibits tumor progression, inhibits tumor fibrosis, inhibits angiogenesis, inhibits cachexia, modulates TME toward an immunostimulatory phenotype, or a combination thereof.
Aspects of the present disclosure are best understood from the following detailed description when read with the accompanying figures. It is noted that, in accordance with the standard practice in the industry, various features are not drawn to scale. In fact, the dimensions of the various features may be arbitrarily increased or reduced for clarity of discussion. The disclosure can be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows:
The following disclosure provides detailed description of many different embodiments, or examples, for implementing different features of the provided subject matter. These are, of course, merely examples and are not intended to limit the invention but to illustrate it. In addition, various embodiments disclosed below may combine or substitute one embodiment with another, and may have additional embodiments in addition to those described below in a beneficial way without further description or explanation. In the following description, many specific details are set forth to provide a more thorough understanding of the present disclosure. It will be apparent, however, to those skilled in the art, that the present disclosure may be practiced without these specific details.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising”, or “includes” and/or “including” or “has” and/or “having” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
In some embodiments, antibodies provided herein have a VH region and VL region respectively including an amino acid sequence having at least about 80% identity, for example, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and any number or range in between, to the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO: 25 and SEQ ID NO:26, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:41 and SEQ ID NO: 42, SEQ ID NO:49 and SEQ ID NO:50, SEQ ID NO:57 and SEQ ID NO:58, SEQ ID NO: 65 and SEQ ID NO:66, SEQ ID NO:73 and SEQ ID NO:74, or SEQ ID NO:81 and SEQ ID NO:82. In other embodiments, antibodies provided herein have a VH region and VL region respectively including an amino acid sequence having at least about 80% identity, wherein the amino acid sequence having at least about 80% identity excludes CDRs.
In some embodiments, the antibody or the antigen-binding portion thereof provided herein includes immunoglobulin framework region (FR) sequences. These sequences are preferably not immunogenic in humans, and are therefore preferably human or humanized FR sequences. Suitable human or humanized FR sequences are known in the art. Specifically preferred FR sequences can be taken from the embodiments shown herein, disclosing the complete antigen binding units and thereby CDR sequences as well as FR sequences.
In some embodiments, antibodies or antigen binding fragments thereof provided herein further include an Fc domain. The term Fc domain refers to an antibody region that includes at least a hinge region, a CH2 domain, and a CH3 domain. In some examples, the Fc domain is an IgG domain, an IgE domain, an IgM domain, and IgD domain, an IgA domain, or an IgY domain. Fc domains of any sequence and from any species can be used, including human, ape, monkey, mouse, rabbit, goat, and others. In some examples, Fc domains are engineered, i.e. non-naturally occurring or recombinant Fc domains generated using techniques of molecular biology, for example. In some examples, the IgG domain is an IgG1 domain, an IgG2 domain, an IgG3 domain, or an IgG4 domain.
In some embodiments, pharmaceutical composition provided herein includes any one of the antibodies or antigen binding fragments thereof provided herein and a pharmaceutically acceptable carrier. In some examples, the pharmaceutically acceptable carrier is conjugated to the C-terminus of one or more polypeptides of the antibody or antigen binding fragment. Any suitable means of conjugating the pharmaceutically acceptable carrier can be used, for example, including covalent conjugation and use of linkers.
In some embodiments, antibody-drug conjugates provided herein can include antibodies or antigen binding fragments thereof provided herein. For example, antibodies or antigen binding fragments thereof that specifically binds to CHI3L1 can be included in antibody-drug conjugates. In some examples, antibody-drug conjugates provided herein include a therapeutic agent, for example, the therapeutic agent includes, but is not limited to chemotherapeutic agent, a therapeutically active compound that inhibits angiogenesis, a signal transduction pathway inhibitor, an immune modulator, an immune checkpoint inhibitor, or a hormonal therapy agent. In some examples, the chemotherapeutic agent includes, but is not limited to gemcitabine, or abraxane.
In some embodiments, antibody-drug conjugates provided herein are used for the treatment of cancer. For example, an antibody of an antibody-drug conjugate targets to an epitope of an antigen on tumor cells, thereby targeting a therapeutic agent included in the antibody-drug conjugate to tumor cells. The therapeutic agent is internalized and released in tumor cells when the antibody-drug conjugate is targeting the tumor cell.
Our previous unpublished data showed that CHI3L1-induced tumor growth and cell proliferation signaling are inhibited by CHI3L1 polyclonal neutralized antibodies in pancreatic, colorectal and lung cancer models. For further clinical application, we developed and characterized phage display-derived fully human monoclonal antibodies (mAb). Here we show that the mAbs targeting CHI3L1 not just inhibit activated AKT and ERK signals, cancer cells migration, tumor progression, tumor fibrosis, angiogenesis and cancer-induced muscle loss (cachexia), but modulate TME toward an immunostimulatory phenotype in vitro and in vivo.
Development of fully human monoclonal antibodies against chitinase-3 like-protein-1 (CHI3L1) with Phage display technology.
To generate antibodies against CHI3L1, selections with an OmniMab phagemid library were carried out. The phagemid library was built up by AP Biosciences Inc. (APBio Inc.) from a collection of peripheral blood mononuclear cells from over a hundred healthy donors.
First round panning was performed using Hyperphage (M13K07ΔρIII, Progen, Heidelberg, Germany). Solution phase panning was used for CHI3L1-specific binder selection and isolation from the OmniMab library. Solution phase panning was performed using recombinant human CHI3L1 which produced by mammalian cell expression system used in first-round selection. Next three rounds enrichment were also used solution phase panning. The specific CHI3L1 binders were screening and isolated by direct ELISA after three and four round panning. Positive binders were isolated and sent for sequencing to confirm the sequence and diversity of the heavy chain. The phage clones recognize human CHI3L1 specifically after three rounds (
These results show that phage clones obtained after three or four rounds of CHI3L1-specific enrichment specifically recognize CH13L1. Positive CHI3L1 binders were renamed according to which round of panning, exclude no binding activity or non-specific binding clones (Table 1).
To quickly screen candidates, the heavy chains and light chains of positive CHI3L1 binders identified by ELISA were amplified, digested and subcloned into an IgG expression vector generated by APBio and carrying the IgG1 constant region. After sequence validation, plasmids were prepared and produced antibody by using mammalian cell expression system. After 6 days of culture, antibody secreted into serum-free medium was affinity purified from culture supernatant by Protein A chromatography. Purified antibody was concentrated, followed by dialysis in PBS buffer. The final concentration of dialyzed protein was determined using a NanoDrop2000 spectrophotometer and the purity and integrity were determined by SDS-PAGE with or without reducing reagent.
CHI3L1 is known to associate with different receptors, such as IL-13Rα2 (He et al. 2013), CRTH2 (Zhou et al. 2015), TMEM219 (Lee et al. 2016), and galectin-3 (Zhou et al. 2015). Recent studies show that CHI3L1 binds to and signals via interleukin-13 receptor alpha 2 (IL-13Rα2) which is significantly upregulated in a number of human cancers (Barderas et al. 2012; Bartolome et al. 2015; Zhao et al. 2015), such as A549 cells, to promote the tumor progression. Therefore, purified antibody leads were functionally screened for their ability to block CHI3L1 binding to IL-13Rα2 on A549 cell line (
Binding and neutralizing ability of fully human mAbs targets against CHI3L1.
CHI3L1 have been reported to interact with several receptors on cancer cells, and trigger downstream signaling for cancer cell growth, proliferation and anti-apoptosis (Kamba et al. 2013; Lee et al. 2016). To investigate the blocking effect of our phage-displayed fully human monoclonal neutralizing antibodies (mAbs) between CHI3L1 and its receptor(s) on cancer cells, we initially screened 11 clones of mAbs for the binding affinity and blockage of CHI3L1-receptor(s) interaction on human lung cancer cell lines (H1299 and A549). His-tagged rCHI3L1 was added for CHI3L1-receptor(s) interaction. Anti-His antibody AHAb with fluorescent F recognized CHI3L1-receptor(s) R complex for detection (left,
Four selected clones of mAbs including R4-4, R4-54, 293R3-2, and 293R3-4 were further tested for blocking effect on human colorectal (HCT116, SW480, and SW620) and pancreatic cancer cell lines (PANC1 and MiaPaCa-2). The rCHI3L1 binding to these cells was significantly reduced by four selected mAbs (
The fully human mAbs inhibit cell growth signaling pathways in different cancer cells.
The involvement of CHI3L1 in intracellular signaling pathways has been reported. For example, CHI3L1 binding to CD44 activates protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling to promote cancer progression (Chen, Jiang et al. 2021). Thus, we tested whether mAb clones R4-4, R4-54, 293R3-2, and 293R3-4 could attenuate AKT and ERK phosphorylation in human pancreatic (PANC1), colorectal (HCT116), and lung (A549) cancer cell lines. PANC1 cells were treated with rCHI3L1 (100 ng/ml) only or rCHI3L1 with different dose of mAbs as indicated for 20 minutes. Immunoblot analysis revealed that four clones of mAb inhibited p-AKT and p-ERK levels induced by rCHI3L1 in a dose dependent manner in PANC1 cells (
Fully human mAbs attenuate growth and migration behaviors in lung, pancreas and colon cancer cell lines.
PI3K/AKT/mTOR signaling pathway is important in regulating cell proliferation, survival and migration (Xu et al. 2020). Therefore, we investigated the effect of four selected mAbs on cancer cell behaviors. Human pancreatic (PANC1) and large cell cancer of the lung (H460) cells were treated with rCHI3L1 only or together with mAbs at two doses, and then subjected to cell counting kit 8 (CCK-8) cell viability assay. Both doses of mAbs treatment reduced the viability of PANC1 and H460 cells (
Since AKT regulates cell migration, we further performed wound healing assay to examine the anti-migratory effect of mAbs on pancreatic (MiaPaCa-2), colorectal (SW480), and lung (A549) cells. We observed that after 16 hours, the wound closure area was increased compared to that in control groups upon rCHI3L1 treatment. Our four selected clones of mAbs significantly reduced cell migration ability induced by rCHI3L1 (
Fully human mAbs show immunostimulatory effects toward immune cells in vitro.
The plasma level of CHI3L1 in various cancer patients is significantly higher than that of healthy controls (Johansen et al. 2004). Besides the direct effect on tumor growth, CHI3L1 also plays roles in modulating tumor microenvironment (TME), such as promoting M2-like tumor associated macrophages (TAM) differentiation and Th1/Th2 inflammatory balance (Kawada et al. 2012). Macrophages rapidly alter their phenotype depending on the external stimuli. Some researches indicated that NF-κB activation triggers M2 polarization (Wang et al. 2014).
To determine the effect of mAbs on immune cells modulation, we investigated the polarization of macrophages and T cell function. We measured NF-κB activity by the secreted embryonic alkaline phosphatase (SEAP) reporter in murine macrophages, RAW-Blue™ cells. The results showed that rCHI3L1-induced NF-κB activity was largely attenuated by four selected mAb clones (
Fully human mAbs mono-treatment exert anti-tumor effects in vivo.
For in vivo verification, four clones of mAb (5 mg/kg) were given to MiaPaCa-2-bearing NOD-SCID mice and compared with gemcitabine (50 mg/kg) for anti-tumor efficacy. Specifically, experimental design of tumor growth inhibition in vivo. MiaPaCa cells (5×106) were injected into pancreas of NOD-SCID mice at Day 0. The mAb clone R4-4, R4-54, 293R3-2, 293R3-4 or control IgG Ab was given by tail-vein injection (i.v.) at indicated time points (Day 7, Day 14, Day 21, and Day 28). Gemcitabine was given through intraperitoneal injection (i.p.) also at indicated time points (Day 7, Day 14, Day 21, and Day 28) and served as a positive drug control. Mice were sacrificed on day 34. All tumors in treatment groups are smaller than IgG control group (
Fully human mAb R4-4 mono-treatment exert anti-tumor and TME ameliorating effects in immunocompetent mice model in vivo.
R4-4 mAb is the only one that could cross-react with murine CHI3L1 (
Fully human mAb R4-54 mono-treatment exert anti-tumor and TME ameliorating effects in humanized mice model in vivo.
Having demonstrated effective CHI3L1 blockade in C57BL/6J immunocompetent mice, we further investigated how fully human monoclonal neutralizing antibody modulated human immune system. We used the advanced severe immunodeficiency (ASID) mice those were implanted with human CD45+ hematopoietic stem cells (HSCs). MiaPaCa-2 and H460 cells were then subcutaneously injected into hHSC reconstituted ASID mice to evaluate the anti-tumor effects of CHI3L1 mAb clone R4-54 on lung and pancreatic cell lines in vivo. IgG isotype or mAb clone R4-54 (5 mg/kg) was given by tail-vein injection every five days, starting on day 14 in MiaPaCa-2 and day 6 in H460-bearing hHSC mice when the tumors reached 300 mm3 in size mimicking the clinical situation of late tumor diagnosis, and mice were sacrificed on day 32 in MiaPaCa-2-bearing hHSC mice and day 27 in H460-bearing hHSC mice. mAb clone R4-54 significantly delayed the growth of MiaPaCa-2 and H460 subcutaneous xenografts (
Fully human mAbs do not show apparent toxicity in treated mice.
Next, we examined whether our mAb clones R4-4 and R4-54 exert any toxicity in vivo. We performed a xenograft model by subcutaneously implanting MiaPaCa-2 cells to NOD-SCID mice. The mice were i.v. injected with IgG isotype, mAb clone R4-4, or clone R4-54 (5 mg/kg) once a week starting from day 7 to day 21, that is, day 7, day 14, and day 21, and mice were sacrificed on day 28. Both R4-4 and R4-54 treatment significantly delayed the growth of MiaPaCa-2 subcutaneous xenografts (
Fully human mAbs and chemotherapy combination treatment exerts synergistic anti-tumor and TME ameliorating effects in vivo.
To come up with the most effective therapeutic strategy, we sought to test whether combined treatment of the first-line chemotherapy gemcitabine and mAb in pancreatic tumor bearing C57BL/6J mice. Murine KPPC-luc cells were orthotopically injected to the pancreas of C57BL/6J, and treatment started on day 14 post-operation. The mAbs clone R4-4 (10 mg/kg or 15 mg/kg), gemcitabine (GEM, 25 mg/kg), Abraxane (2.5 mg/kg) or control IgG Ab were given by i.p. at indicated time point (day 14, day 20, and day 25), and mice were sacrificed on day 29. KPPC-luc bearing mice was further divided into six groups, including IgG isotype, gemcitabine (25 mg/kg), R4-4 (10 or 15 mg/kg), gemcitabine (25 mg/kg) plus R4-4 (10 mg/kg), or gemcitabine (25 mg/kg) plus Abraxane (2.5 mg/kg). The last group, GEM plus Abraxane, is the clinically use combination chemotherapy used for pancreatic cancer treatment. Luciferase activity of tumor cells and tumor weight measurement showed obvious restricted tumor growth in GEM plus R4-4 which was comparable to the GEM plus Abraxane treated groups (
Since pancreatic patients often suffer from cachexia, the cancer-related muscle loss disorder (Henderson et al. 2018; Yoo et al. 2021), we further performed the mouse muscle fiber area analysis in IgG and R4-4 (10 mg/kg) groups. The muscle fiber area measurement, although were heterogeneous (
ForteBio binding analysis between anti-CHI3L1 mAbs and rCHI3L1.
In addition to the functionality validation is important during the early stages of antibody discovery. It is also important to have an efficient and accurate way to assess the success or failure of a particular discovery effort. First, binding affinity to the target is a critical criterion for the success of the antibody. Second, epitopic coverage is also important during the very early stages of discovery, for without targeting the relevant epitope.
Here, four candidates as showed above were conducted for affinity determination by biolayer interferometry analysis (ForteBio, USA). Anti-CHI3L1 mAbs were loaded at 5 μg/mL on AHC (Anti-Human IgG Fc Capture, ForteBio, Cat #18-5060) for signal reach to approximate 0.5 nm. Sensors then were exposed to human CHI3L1 extracellular domain fused with flag and His tag recombinant protein, hCHI3L1/flag-His, solutions at 10 nM with 2 time serial dilution for 7 concentrations. Measurement were performed as follows: 1 min baseline in kinetic buffer, 5 minute association in 7 different concentrations hCHI3L1/flag-His and 5 minute dissociation into kinetic buffer. The kinetic parameters were calculated and produced by Octet Data Acquisition and Analysis Software. In this assay, the binding affinity of anti-CHI3L1 antibodies against recombinant hCHI3L1 fused with flag-His tag were around 1.13 E-09 M to 4.22E-09 M (
Mapping/Binning of the epitopes of the four monoclonal antibodies.
In order to know the epitopic coverage for those four antibodies to increase the success of therapeutic CHI3L1 antibody development, epitope binning is used to characterize the binding site of mAbs to the corresponding antigen and summarized in the Table 4. In epitope binning, mAbs specific to the same target protein are tested pairwise against all mAbs in a set to assess whether they block one another's binding to a specific site of the antigen or not. The mAbs that block binding to the same epitope are “binned” together. mAbs within the same bin often function similarly, so epitope bins can narrow down the choices to fewer candidates for investigators to choose from. The in tandem epitope binning assay conducted on Octet Red96 comprised a five-step binding cycle; 1) a buffer baseline was established for 30 sec, 2) 100 nM hCHI3L1/flag-His was captured for 5 min, 3) 100 nM mAb array was loaded to saturate the captured antigen for 10 min, 4) 100 nM of the test mAb was bound for 5 min, and 5) the capture surfaces were regenerated for 30 sec. Anti-His surfaces were regenerated with 10 mM glycine pH 1.7. Raw data was processed using ForteBio's Data Analysis HT Software 11.1 and the antibody pairs were assessed for competitive binding. Additional binding by the second antibody indicates an unoccupied epitope (non-competitor, marked as *c in Table 4), while no binding indicates epitope blocking (competitor, marked as *b in Table 4) and self-competing antibodies were marked as *a in Table 4. Among four anti-CHI3L1 mAbs, there are two epitope clusters. R4-4 was identified as an exclusive epitope. R4-54, 293R3-2 and 293R3-4 had unidirectional blocking results which occur with disparate-affinity antibodies of closely adjacent or minimally overlapping epitopes.
While the disclosure has been described by way of example(s) and in terms of the preferred embodiment(s), it is to be understood that the disclosure is not limited thereto. On the contrary, it is intended to cover various modifications and similar arrangements and procedures, and the scope of the appended claims therefore should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements and procedures.
This application is the United States national phase of International Application No. PCT/US2022/077374, filed Sep. 30, 2022, and claims priority to U.S. Provisional Patent Application No. 63/272,701, filed Oct. 28, 2021, the disclosures of which are hereby incorporated by reference in their entireties. The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the XML file containing the Sequence Listing is NP-30914-US_SEQ_LIST.xml. The size of the XML file is 80,213 bytes, and the XML file was created on Feb. 17, 2024.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/77374 | 9/30/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63272701 | Oct 2021 | US |
