TW201632202A - Treatment of breast cancer - Google Patents

Abstract

The invention relates, in part, to a method of selectively treating a patient having breast cancer, comprising administering a therapeutically effective amount of a M-CSF antagonist to the patient on the basis of the patient having a level of CD163 expression that is predictive that the patient will respond to an M-CSF antagonist.

Description

Breast cancer treatment

The present invention relates to methods of treatment and methods of screening for patients suffering from breast cancer treated with M-CSF antagonists.

Breast cancer is the most common cancer among women worldwide. It is estimated that there were 1.38 million new cases in 2008, and it is also the most common cause of cancer death in women, with 458,000 deaths. Triple negative breast cancer (TNBC) accounts for approximately 15% of newly diagnosed breast cancer (25% of metastatic breast cancer) and is characterized by a lack of estrogen (ER) and progesterone (PR) receptor expression and lack of human epidermal growth factor receptor Excessive performance of 2 (HER2).

Currently, there is no target therapy for this heterogeneous breast cancer subtype and the only treatment option is chemotherapy. Although several studies have shown that TNBC disease is sensitive to chemotherapy, the prognosis is still poor, and its metastatic potential is shorter than the non-TNBC disease after initial treatment, and the clinical course is more aggressive. Most patients in adjuvant applications receive anthracycline-based and taxane-based chemotherapy, and there are no further care therapies for patients with metastatic TNBC. However, emerging data show that platinum salts (i.e., cisplatin and carboplatin) and gemcitabine and/or combinations thereof are highly active in early and late TNBC and are therefore widely used in clinical applications. The median survival of metastatic TNBC is about 1 year, making TNBC a disease with high unmet medical needs.

TNBC and generally breast cancer are heterogeneous, and patients are not necessarily in a similar manner A similar drug regimen response. Therefore, the use of markers that predict drug response for stratification of patients with breast cancer disease will help clinicians screen and establish effective treatment strategies. In addition, patient stratification will avoid safety issues and the high economic burden associated with long-term therapy. Therefore, there is a need to develop methods of treating breast cancer diseases with therapeutic agents that first determine the patients most likely to benefit from the therapeutic agent.

Novel treatments for patients with breast cancer are provided herein. In particular, the invention relates to the use of a macrophage colony stimulating factor (M-CSF) antagonist, such as the H-RX1 antibody, as described herein, for the treatment of breast cancer, such as triple negative breast cancer (TNBC). The invention further encompasses the use of an M-CSF antagonist, such as an H-RX1 antibody, as described herein in a breast cancer population prior to treatment with a M-CSF antagonist to identify a patient who is likely to respond favorably. Personalized therapy that maximizes and minimizes risk. In particular, the invention includes, for example, identifying a patient with breast cancer having a predetermined level of tumor-associated macrophage (TAM), such as a triple-negative breast cancer patient, by analyzing the CD163 expression characteristic of a subset of TAM-derived TAMs. . CD163 expression in tumor samples obtained from triple-negative breast cancer patients, for example, mRNA expression and/or protein levels or densities can be used to indicate whether the patient is more likely to respond favorably to M-CSF antagonist therapy.

In one aspect, the invention includes a method of treating a patient having triple negative breast cancer comprising selectively administering to the patient a therapeutically effective amount of an M-CSF antagonist. In one embodiment, the M-CSF antagonist is an antibody or antigen-binding fragment thereof. The M-CSF antibody or antigen-binding fragment thereof can comprise a heavy chain variable region comprising a CDR1, CDR2 and CDR3 domain; and a light chain variable region comprising a CDR1, CDR2 and CDR3 domain, wherein the heavy chain variable region CDR3 comprises The amino acid of the sequence depicted in SEQ ID NO: 7; and the light chain variable region CDR3 comprise an amino acid having the sequence depicted as SEQ ID NO: 10. The antibody or antigen binding portion thereof can bind to human M-CSF having a binding affinity of about 10 ^-7 M.

In another aspect, the invention includes a method of treating a patient suffering from breast cancer, eg, triple-negative breast cancer, comprising selectively administering a therapeutically effective amount of an M-CSF antagonist, eg, an M-CSF antibody or An antigen binding fragment, for example, an antibody or a fragment thereof having the CDR or H-RX1 as shown in Table 1.

In another aspect, the invention comprises a method of treating a patient afflicted with breast cancer, eg, triple-negative breast cancer, comprising selectively administering to a patient based on a patient having a TAM density that predicts a likelihood of responding to an M-CSF antagonist A therapeutically effective amount of an M-CSF antagonist, for example, an M-CSF antibody or antigen-binding fragment thereof, for example, H-RX1, is administered.

In another aspect, the invention comprises a method of treating a patient afflicted with breast cancer, eg, triple-negative breast cancer, comprising selectively providing a patient based on a CD163 performance level predicting its likely response to an M-CSF antagonist The patient is administered a therapeutically effective amount of an M-CSF antagonist, for example, an M-CSF antibody or antigen-binding fragment thereof, for example, H-RX1.

In another aspect, the invention comprises a method of treating a patient suffering from breast cancer comprising selectively administering to the patient a therapeutically effective amount based on the patient having a CD163 protein density predictive of its potential response to the M-CSF antagonist An M-CSF antagonist, for example, an M-CSF antibody or antigen-binding fragment thereof, for example, comprising CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6), and CDR3 (SEQ ID NO: 7) A heavy chain variable region and an antibody or binding fragment comprising a CDR1 (SEQ ID NO: 8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) light chain variable region.

In still another aspect, the invention includes a method of selectively treating a patient suffering from breast cancer comprising: based on a patient having a CD163 performance level predicted to be likely to respond to M-CSF compared to a control group (eg, A CD163 is above the threshold of cut-off threshold to screen for treatment with a therapeutically effective amount of a M-CSF antagonist; and thereafter, a therapeutically effective amount of an M-CSF antagonist is administered.

In yet another aspect, the invention comprises a method of screening a patient for breast cancer treated with an M-CSF antagonist, comprising: based on the patient having a prediction compared to a control group It is possible to screen for a patient treated with a therapeutically effective amount of an M-CSF antagonist in a CD163 performance level (eg, above the cut-off threshold) for M-CSF antagonist response.

In still another aspect, the invention comprises a method of selectively treating a patient suffering from breast cancer, comprising: analyzing an increase in CD163 performance level of a patient's biological sample; and thereafter, selectively administering to the patient any of the following One: based on the patient's CD163 expression level (eg, mRNA or protein) predicted to be likely to respond to M-CSF compared to the control group, for example, the sample has an improved CD163 performance level compared to the control group, administration A therapeutically effective amount of an M-CSF antagonist; or based on a patient's biological sample that does not have an increased level of CD163 expression compared to a control group in order to predict its likely response to an M-CSF antagonist, administering a therapeutically effective amount in addition to M-CSF Antagonist therapy.

In still a further aspect, the invention comprises a method of selectively treating a patient suffering from breast cancer comprising analyzing a CD163 performance level of a patient biological sample; and thereafter, based on the patient biological sample having a prediction that it is possible for M- CD163 expression level of the CSF response (eg, the sample has an increased level of CD163 expression or presence compared to the control group) to screen for treatment with a therapeutically effective amount of the M-CSF antagonist; and thereafter, selected The patient is administered an M-CSF antagonist.

In still another aspect, the invention comprises a therapeutically effective amount of an M-CSF antagonist for use in treating a patient suffering from breast cancer, characterized in that the therapeutically effective amount of an M-CSF antagonist is based on the patient having The patient was enrolled in the CD163 performance level predicted to be likely to respond to the M-CSF antagonist compared to the control group.

In yet another aspect, the invention comprises a therapeutically effective amount of an M-CSF antagonist for use in treating a patient suffering from breast cancer, characterized in that the patient has a predictive response to an M-CSF antagonist based on the patient's prediction CD163 expression level (eg, compared to a control group, eg, (above the threshold of cut-off threshold)) to screen for a patient treated with an M-CSF antagonist; and thereafter, administering a therapeutically effective amount of M- CSF antagonist.

In another aspect, the invention includes a method for predicting a patient suffering from breast cancer A method of treating a therapeutically effective amount of an M-CSF antagonist for the likelihood of a response comprising analyzing a CD163 performance level of a patient's biological sample, wherein the CD163 performance level compared to the control group indicates that the patient will be treated with an M-CSF antagonist The increased likelihood of response; and the absence of CD163 performance compared to the control group indicates a decrease in the likelihood that the patient will be treated with an M-CSF antagonist.

In still another aspect, the invention includes a method of selectively treating a patient suffering from breast cancer, eg, triple-negative breast cancer, comprising predicting that it is likely to respond to an M-CSF antagonist based on the patient having a comparison with a control group The CD163 exhibits a level of selectivity to the patient to i) a combination of i) a therapeutically effective amount of an M-CSF antagonist, and ii) a therapeutically effective amount of a second agent (e.g., carboplatin or gemcitabine). In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In still another aspect, the invention includes a method of selectively treating a patient suffering from breast cancer, eg, triple negative breast cancer, comprising: based on the patient having a predicted likelihood of responding to M-CSF compared to a control group CD163 expression level for screening for a therapeutically effective amount of an M-CSF antagonist, and ii) a therapeutically effective amount of a second agent, eg, a chemotherapeutic agent, such as a combination of carboplatin or gemcitabine; and thereafter Simultaneously, separately or sequentially, i) a combination of a therapeutically effective amount of an M-CSF antagonist, and a therapeutically effective amount of a second agent. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In yet another aspect, the invention comprises a method of selectively treating a patient suffering from breast cancer, comprising selectively selecting based on a patient having a level of CD163 expression predicting a likelihood of responding to M-CSF compared to a control group The patient is administered a therapeutically effective amount of an M-CSF antagonist, carboplatin, and gemcitabine. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In yet another aspect, the invention comprises a method of selectively treating a patient suffering from breast cancer comprising screening for a CD163 performance level based on a patient's predicted likelihood of responding to M-CSF compared to a control group A patient treated with a therapeutically effective amount of an M-CSF antagonist, carboplatin, and gemcitabine; and thereafter, a therapeutically effective amount of a M-CSF antagonist, carboplatin, and gemcitabine is administered simultaneously, separately, or sequentially. The combination. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In still another aspect, the invention includes a method of selectively treating a patient suffering from breast cancer, comprising analyzing a CD163 protein level of a patient's biological sample; and thereafter, based on the patient biological sample having a predicted comparison to the control group The CD163 expression level likely to respond to M-CSF is screened for patients treated with M-CSF antagonists; and thereafter, M-CSF antagonists are administered to selected patients. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

It can be selected by any known technique, for example, from Northern blot analysis, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), immunoassay, immunohistochemistry (IHC). , ELISA and Western blotting techniques to detect CD163. In particular, CD163 protein can be detected by quantifying IHC, for example, by immunostaining CD163 and by quantifying CD163 protein performance using an automated image analysis instrument.

The administration step of any of the above technical means may comprise administering to the patient a dose between about 5 and 30 mg/kg at time 0, for example, a 10 mg/kg M-CSF antagonist ("first administration" 7 to 14 days after the first administration, for example, 8 mg/kg of M-CSF antagonist ("extra dose"), and then every 3 weeks after the first administration, for example 10 mg/kg of M-CSF antagonist. In a specific embodiment, the M-CSF antagonist is a heavy chain variable region comprising a CDR1, CDR2 and CDR3 domain; and comprising CDR1, CDR2 and An antibody or antigen-binding fragment thereof, wherein the heavy chain variable region CDR3 comprises an amino acid having the sequence depicted in SEQ ID NO: 7; a sequence of amino acid light chain variable region CDR3, and wherein the antibody or fragment thereof is administered to a patient at a dose of 10 mg/kg on the first administration, 7 to 14 days after the first administration For example, an additional dose of 10/mg/kg is administered at a dose of 10 mg/kg for 8 days, and then the antibody or fragment thereof is administered at a dose of 10 mg/kg every three weeks after the first administration. Alternatively, gemcitabine and carboplatin may be administered to the patient on the first day and every three weeks thereafter. In one embodiment, the therapeutic agent can be administered intravenously, sequentially, simultaneously or separately.

In another embodiment, in the methods described above, the M-CSF antagonist is an M-CSF antibody or antigen-binding fragment thereof. The M-CSF antibody may comprise 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In another embodiment, in the methods described above, the M-CSF antibody or fragment comprises a VH comprising SEQ ID NO: 2 and a VL comprising SEQ ID NO: 4, or having 97 to 99% identical Amino acid sequence.

In yet another aspect, the invention includes a method of making information for predicting the responsiveness of a patient with breast cancer to a responsiveness to treatment with an M-CSF antagonist, comprising: analyzing a CD163 performance level of a patient's biological sample; And recording the results of the analysis step on a physical or non-physical media form suitable for dissemination, wherein if the result is that the CD163 performance level is increased compared to the control group (eg, the level is above a predetermined threshold level), then the patient is instructed to There is an increased likelihood of a therapeutic response involving an M-CSF antagonist. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In another embodiment, a method of treating a patient having breast cancer, eg, triple-negative breast cancer, comprising: administering a therapeutically effective amount of an M-CSF antagonist and treatment simultaneously, separately, or sequentially An effective amount of a combination of gemcitabine and carboplatin. In one implementation In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In yet another embodiment, a method of selectively treating a patient suffering from breast cancer, comprising: a) analyzing a CD163 protein level of a patient's biological sample; b) predicting that the patient is likely to be CD163 expression level of the M-CSF response to screen for treatment with a combination of i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii) a therapeutically effective amount of a third agent And c) thereafter, simultaneously, separately or sequentially, i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii) a therapeutically effective amount A combination of three agents. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In yet another embodiment, a method of selectively treating a patient suffering from breast cancer, comprising: a) analyzing a tumor-associated macrophage (TAM) level of a patient's biological sample; b) based on the patient having a phase with the control group Comparing the level of TAM content that is likely to respond to M-CSF to screen for a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii) a therapeutically effective amount of a third agent a patient treated with a combination of agents; and c) thereafter, simultaneously, separately or sequentially, i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii a combination of a therapeutically effective amount of a third agent. In some embodiments, the breast cancer is TNBC. In some embodiments, the CD163 protein expression level is above a threshold level, for example, each biopsy slice as described herein is determined to be at least TAM density measured by quantitative immunoassay for CD163+ pixel density. 8%, 10%, 15%, 20%, 30% or 40%. In still other embodiments, each biopsy slice as described herein is determined to be less than a TAM density by measuring the CDM+ pixel density measured by a quantitative immunoassay using a threshold or control level. 8%. In one embodiment, the M-CSF antagonist is an antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In various embodiments, a method of the above, wherein the M-CSF antagonist is an M-CSF antibody or fragment thereof. In related embodiments, the antibody or fragment thereof comprises 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1.

In still other embodiments, the above method is provided wherein the second agent is carboplatin and the third agent is gemcitabine.

In another embodiment, a method of selectively treating a patient suffering from TNBC is provided, comprising: a) analyzing a CD163 protein level of a patient's biological sample; b) predicting that it is likely to be M based on the patient compared to the control group a CD163 expression level of a CSF response to screen a patient for treatment with a combination of i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii) a therapeutically effective amount of a third agent; And c) thereafter, i) administering a therapeutically effective amount of the M-CSF antagonist, ii) a therapeutically effective amount of the second agent, and iii) a therapeutically effective amount of the third agent simultaneously, separately or sequentially. a combination of agents; wherein the M-CSF antagonist is an M-CSF antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1, and wherein The second agent is carboplatin and the third agent is gemcitabine.

In yet another embodiment, a method of selectively treating a patient suffering from TNBC is provided, comprising: a) analyzing a TAM of a patient's biological sample; b) predicting that it is likely to be M-based based on the patient compared to the control group The level of TAM content of the CSF response (eg, above the cut-off threshold) to screen for a therapeutically effective amount of the M-CSF antagonist, ii) a therapeutically effective amount of the second agent, and iii) a therapeutically effective amount a patient treated with a combination of three agents; and c) thereafter, simultaneously, separately or sequentially, i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and Iii) a therapeutically effective amount of a third agent combination; wherein the M-CSF antagonist is an M-CSF comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. An antibody or fragment thereof, wherein the second agent is carboplatin and the third agent is gemcitabine.

In another aspect, the invention encompasses a therapeutically effective amount of M-CSF antagonism A pharmaceutical composition of a combination of a drug, carboplatin and gemcitabine.

In still another aspect, the invention includes a pharmaceutical composition comprising an M-CSF antagonist suitable for simultaneous, separate or sequential administration to treat TNBC. The M-CSF antagonist can be an M-CSF antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. In one embodiment, the M-CSF antagonist is H-RX1.

In yet another aspect, the invention comprises a pharmaceutical composition comprising a therapeutically effective amount of a combination of an M-CSF antagonist, carboplatin and gemcitabine, which is suitable for simultaneous, separate or sequential administration. With the treatment of TNBC. The M-CSF antagonist can be an M-CSF antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. In one embodiment, the M-CSF antagonist is H-RX1.

In another aspect, the invention provides a commercially available package comprising a therapeutic agent M-CSF antagonist, carboplatin and gemcitabine, together with simultaneous, separate or sequential administration in the treatment of TNBC Description. The M-CSF antagonist can be an M-CSF antibody or fragment thereof comprising 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. In one embodiment, the M-CSF antagonist is H-RX1.

a) definition

In order to more readily clarify the invention, specific terms are first defined. Other definitions are described in the detailed description.

The term "antibody" as used herein refers to an entire antibody that interacts with the M-CSF epitope and inhibits signal transduction (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution). A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and V _L regions can be further subdivided into regions called complementarity determining (CDR) of the hypervariable regions, called the dispersed more conserved framework regions (FR) of the inside thereof. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise a binding domain that interacts with the antigen. The constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fv (scFv), disulfide-linked Fv (sdFv), Fab fragments, F(ab'). A fragment and an anti-genotype (anti-Id) antibody (including, for example, an anti-Id antibody against an antibody of the present invention), and an epitope-binding fragment of any of the above. Such antibodies can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), classes (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclasses.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. At this point, it is understood that the variable domain of both the light (VL) and heavy (VH) chain portions determines antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. According to the protocol, the numbering of the constant region domain increases as it becomes further away from the antigen binding site or the amine terminus of the antibody. The N-terminus is a variable region and the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy terminus of the heavy and light chains, respectively.

The phrase "antibody fragment" as used herein refers to the ability of an antibody to retain (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution) to specifically interact with the M-CSF epitope and to inhibit One or more parts of the message. Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab) ₂ fragments, two Fab fragments comprising a double sulfur bridge linked in the hinge region a valency fragment; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of one arm of the antibody; a dAb fragment (Ward et al . (1989) Nature 341:544-546), which is derived from the VH domain Composition; and singular complementarity determining regions (CDRs). Moreover, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be recombined by making them a VL and VH block pair to form a synthetic linker of a single protein chain of a monovalent molecule. Linkage (known as single-chain Fv (scFv); see, for example, Bird et al , (1988) Science 242: 423-426; and Huston et al, (1988) Proc. Natl. Acad. Sci. 85: 5879- 5883). Such single chain antibodies are also intended to be encompassed by the term "antibody fragment". Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and useful fragments are screened in the same manner as intact antibodies.

The phrase "specifically (or selectively) binds" to an antibody (eg, an M-CSF-binding antibody) refers to determining the binding reaction of a homologous antigen (eg, human M-CSF) in a heterogeneous population of proteins and other biological agents. . In addition to the equilibrium constant (K _A ) described above, the M-CSF-binding antibody of the present invention generally also has less than 5 × 10 ^-2 M, less than 10 ^-2 M, less than 5 × 10 ^-3 M, and less than 10 ^{- 3} M, less than 5 × 10 ^-4 M, less than 10 ^-4 M, less than 5 × 10 ^-5 M, less than 10 ^-5 M, less than 5 × 10 ^-6 M, less than 10 ^-6 M, less than 5 × 10 ^{- 7} M, less than 10 ^-7 M, less than 5 × 10 ^-8 M, less than 10 ^-8 M, less than 5 × 10 ^-9 M, less than 10 ^-9 M, less than 5 × 10 ^-10 M, less than 10 ^-10 M , less than 5 × 10 ^-11 M, less than 10 ^-11 M, less than 5 × 10 ^-12 M, less than 10 ^-12 M, less than 5 × 10 ^-13 M, less than 10 ^-13 M, less than 5 × 10 ^-14 M a dissociation rate constant (K _D ) (k _off /k _on ) of less than 10 ^-14 M, less than 5 × 10 ^-15 M or less than 10 ^-15 M or less, and binding to a non-specific antigen ( For example, HSA has at least twice the affinity for binding to M-CSF. In one embodiment, the antibody or fragment thereof has, as assessed using methods described herein or known to those skilled in the art (eg, BIAcore analysis, ELISA, FACS, SET) (Biacore International AB, Uppsala, Sweden). Dissociation constants (K _d ) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 10 pM, less than 1 pM. The term "K _assoc " or "K _a " as used herein refers to the association rate of a particular antibody-antigen interaction, and the term "K _dis " or "K _d " as used herein refers to a specific antibody-antigen. The rate of dissociation of the interaction. The term "K _D" as used herein refers to the dissociation constant, K _d from its line and K _a ratio (i.e., K _d / K _a) obtained and expressed as molarity (M). K _D values for antibodies of the present methods may be used by well established in the art to measured. A method of measuring K _D by using antibody-based surface plasmon resonance, or using the biosensor system such as a Biacore ^® system.

An "M-CSF antagonist" as used herein may be an antibody or other antigen binding protein, a small molecule, a nucleic acid (such as siRNA) or any other such molecule that interferes with M-CSF activation or function. In one example, the M-CSF antagonist is an antibody that binds to M-CSF. In a specific embodiment, the M-CSF antagonist binds to M-CSF and neutralizes the biological activity of M-CSF signaling. Antibodies that inhibit M-CSF activity can achieve a statistically significant reduction in such measured parameters by at least 10%, by at least 50%, 80%, or 90%, and in certain embodiments, the antibodies of the invention can be inhibited Greater than 95%, 98% or 99% of M-CSF functional activity.

As used herein, the phrase "therapeutically effective amount" means a therapeutic or prophylactic M-CSF antagonist, such as an antibody, which is suitable for use in embodiments of the invention, when administered according to a desired therapeutic regimen. The amount of the desired therapeutic or prophylactic effect or response will be exerted.

Human "M-CSF" as used herein refers to having with Kawasaki et al, Science 230: 291 (1985), Cerretti et al, Molecular Immunology, 25: 761 (1988) or Ladner et al, EMBO Journal 6: 2693 Human polypeptides of substantially identical amino acid sequences of mature human M-CSF alpha, M-CSF beta or M-CSF gamma polypeptides as described in (1987), each of which is incorporated herein by reference. The term reflects the understanding that the three mature M-CSFs have different amino acid sequences as described above, and that the active form of M-CSF is a disulfide-bonded dimer; therefore, the term "M-CSF" "In the case of a biologically active form, it means a dimeric form." "M-CSF dimer" means dimerized and includes not only homodimers (consisting of two identical types of M-CSF monomers) but also heterodimerization Two M-CSF polypeptide monomers of the body (consisting of two different monomers). The M-CSF monomer can be converted to the M-CSF dimer in vitro as described in U.S. Patent No. 4,929,700, the disclosure of which is incorporated herein by reference.

The phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to M-CSF is substantially free of specific binding to M-CSF). An antibody other than the antigen). However, an isolated antibody that specifically binds to M-CSF may have cross-reactivity with other antigens. Moreover, the isolated antibodies may be substantially free of other cellular material and/or chemicals.

The phrase "conservatively modified variant" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, a conformationally modified variant system refers to a nucleic acid that encodes the same or substantially the same base acid sequence, or in which case the nucleic acid does not encode an amino acid sequence, encoded into substantially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode amino acid alanine. Thus, at each position where alaline is specifically identified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. These nucleic acid changes are "silent changes", which are one species that undergo a change in conservation modification. Each nucleic acid sequence encoding a polypeptide herein also describes every possible silent change in the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (other than the AUG, which is typically the only codon for methionine and the TGG, which is typically the only codon for tryptophan), can be modified to produce functionally identical Molecule. Thus, each silent change in the encoded polypeptide of a nucleic acid is implicit in each of said sequences.

The term "cross-competition" is used herein to mean the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to M-CSF in a standard competitive binding assay.

Standard competitive binding assays can be employed to determine whether an antibody or other binding agent can interfere with the ability or extent of binding of another antibody or binding molecule to M-CSF and thus determine whether it can be considered cross-competing in accordance with the present invention. A suitable analytical method for using Biacore technology (For example, by using a BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), it is possible to measure the degree of interaction using surface plasmon resonance techniques. Another assay for measuring cross-competition uses an ELISA-based approach.

The phrase "percent identity" refers to the same two or more sequences or subsequences in the context of two or more nucleic acid or polypeptide sequences. When the maximum correspondence on the comparison and comparison window is specified or specified as a subsequent sequence comparison algorithm or by artificial alignment and visual inspection, if the two sequences have the same ratio of the same amine group An acid residue or nucleotide (i.e., on a particular region, or when not specified over the entire sequence, has 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90) %, 95%, or 99% consistency), the two sequences are "substantially the same." Optionally, the length is in the region of at least about 50 nucleotides (or 10 amino acids) or more preferably in the range of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or There is consistency in the area of more amino acids).

In the case of sequence comparisons, usually one sequence is used as a reference sequence for comparison with the test sequences. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the sequence coordinates are specified, and the sequence algorithm program parameters are specified. Preset program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the program parameters.

The term "individual" includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "individual" are used interchangeably herein except as noted.

"Tumor" refers to the growth and proliferation of tumor cells (whether malignant or benign), and all precancerous and cancerous cells and tissues.

Unless otherwise indicated herein, the term "about" in relation to the value x means +/- 10%.

The term "analysis" is used to refer to the act of identifying, screening, detecting, testing, measuring or determining, and the behavior can be performed by any method. For example, IHC, RT-PCR, ELISA assay, Northern blot, immunohistochemistry, Western blot, mass spectrometry, etc. can be used to analyze the CD163 performance level of the sample. The term "detection" (and similar) means the act of extracting specific information from a given source, either directly or indirectly. The terms "analyze" and "determine" encompass the conversion of a substance, for example, a biological sample, such as a blood sample or other tissue sample, by conversion of one state to another state by physical testing of the sample.

The term "obtaining" means taking in any method, for example, by physical intervention (eg, biopsy, blood draw) or non-physical intervention (eg, by transmitting information through a server), for example, acquiring possession.

The phrase "analyze biological sample..." and the like means the increase or absence of the presence or presence of a CD163 nucleic acid expression, protein or activity of a testable sample (directly or indirectly). It should be understood that where the presence or absence of a substance indicates a probability and the absence of a substance indicates a different probability, the presence or absence of such a substance can be used to guide treatment decisions. For example, whether a patient is determined by determining the presence of an increased CD163 indicates that the patient will be at a protein level of CD163 in a patient sample that is more likely to respond to treatment with an M-CSF antagonist or a combination of an M-CSF antagonist and another agent Has an increased CD163 protein performance.

Unless otherwise indicated, the term "derivative" is used to define an amino acid sequence variant and an M-CSF antagonist, eg, a covalent modification of an M-CSF antibody or antigen binding portion thereof (eg, PEGylation) Pegylation), deamidamine, hydroxylation, phosphorylation, methylation, etc.). A "functional derivative" includes a molecule having the same qualitative biological activity as the disclosed M-CSF antagonist, for example, an M-CSF binding molecule. Functional derivatives include fragments of M-CSF antagonists and peptide analogs as disclosed herein.

The phrase "substantially identical" amino acid or nucleotide sequence-related (e.g., V _H or V _L domain) will have the same mean specific differences as compared to the reference sequence or reference sequence specific insubstantial (e.g., by Conservative amino acid substitution). No substantial differences in the amino acid sequence consists of five specific regions (e.g., V _H or V _L domain) of amino acids in minor changes, such as at 1 or 2 substituent (e.g., conserved substitutions, such as Su-amine Acid exchange for serine, or substitution at a position that does not involve antibody activity, structural integrity, complement binding, etc.). In the case of antibodies, the second antibody has the same specificity and has at least 50% affinity. Sequences that are substantially identical (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity of the derivative M-CSF antibody can be about 90% or greater, for example, 90%, 91%, 92%, 93%, 94%, compared to the disclosed sequences. 95%, 96%, 97%, 98%, 99% or higher.

The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient.

The term "administering" in connection with a compound or antibody, eg, an M-CSF antibody or antigen-binding fragment thereof, or another agent, is used to mean delivery of the compound or antibody to a patient by any route.

The term "treatment" or "treat" refers to both prophylactic or preventive treatment (as the case may be) and curative or disease-modifying treatment, including in the case of an infectious disease or a suspected infected disease. Treatment of patients at risk and patients who are ill or have been diagnosed with a disease or medical condition, and include inhibition of clinical relapse. Treatment may be administered to a patient having a medical condition or ultimately suffering from the disease to prevent, cure, delay the onset of one or more symptoms of the disease or relapsed disease, reduce the severity of one or more symptoms of the disease or relapsed disease, or ameliorate the disease Or one or more symptoms of a recurrent disease, or to prolong the survival of a patient beyond what is expected without such treatment.

The use of the phrase "responding to a treatment" means delivering a particular treatment, for example, a patient after the M-CSF antibody or fragment thereof exhibits a clinically meaningful benefit from the treatment. The phrase "reaction to treatment" is intended to be interpreted in comparison rather than as an absolute response. For example, forecast Breast cancer patients with higher levels of CD163 markers than the control group benefited more from treatment with M-CSF antagonists than breast cancer patients who did not have increased CD163 levels (nucleic acids, eg, mRNA or protein). In another example, a breast cancer patient has a CD163 marker that exceeds a certain threshold level. For example, an IHC analysis indicates that the level of the CD163 marker exceeds a certain threshold, for example, exceeds 15%, and the cancer patient is predicted to use M-CSF. Antagonist treatments have a greater benefit than breast cancer patients whose levels of CD163 do not exceed the threshold (nucleic acids, eg, mRNA or protein).

The use of the phrase "receiving data" means obtaining information by any available means, for example, verbally, electronically (e.g., by email, encoded on a floppy disk or other medium), writing, and the like.

As used herein, the use of "screening" and "screening" in the context of a patient means that a particular patient is based on (due to) a particular patient having predetermined criteria, eg, the patient has an increased level of CD 163 (eg, nucleic acid, eg, The mRNA or protein) or CD163 that exceeds a predetermined level of margin is specifically selected from a larger group of patients. Similarly, "selective treatment" refers to the treatment of a patient with a particular condition, wherein the patient is specifically selected from a larger group of patients based on a particular patient having predetermined criteria, for example, because the patient has an increased level of CD163 A breast cancer patient who is specifically selected for treatment with protein. Similarly, "selective administration" refers to a particular choice based on (due to) a particular patient having predetermined criteria, for example, a patient having an increased level of CD163 protein and a patient selected from a larger group of patients. drug. By screening, selective treatment, and selective administration is meant that a personalized therapy is administered to a patient based on the particular biology of the patient, rather than a standard treatment regimen based solely on patients with a particular disease. Screening, as used herein, does not refer to a patient's occasional treatment with an increased level of CD163 expression, but rather a CD163 expression based on the level of likelihood that the patient is likely to respond to M-CSF compared to the control group. Preference is given to administering M-CSF antagonists to patients. Therefore, selective treatment is different from standard treatment, and selective treatment is given to specific patients to all patients, and with CD163 Performance levels or protein levels are irrelevant.

In various embodiments, the patient is screened for treatment based on the inclusion criteria. For example, criteria may include, but are not limited to, adult women with advanced TNBC ( 18 years old); based on the last available tumor tissue, estrogen-receptor negative (ER-), progesterone receptor negative (PgR-) and human epidermal growth factor-2 receptor negative (HER2-) BC Histological or cytological evidence for laboratory testing; pre-treatment tumor biopsy confirming high TAM levels; radiological or objective evidence of recurrence or development before treatment; with at least one detectable lesion, including osteolytic or mixed, according to RECIST 1.1 Sexual (osteolytic + osteogenic) bone lesions with soft tissue components that meet measurable criteria) and/or bone lesions in the absence of detectable disease (undetectable osteolytic or mixed (osteolytic + adult) Patients with bone).

As used herein, "predicting" indicates that the methods described herein provide information to enable a health care provider to determine that an individual having breast cancer will respond to treatment with an M-CSF antagonist, such as an M-CSF antibody or antigen-binding fragment, or The possibility of treating the response with an M-CSF antagonist such as an M-CSF antibody or antigen-binding fragment will be more advantageous. It does not refer to the ability to predict the response with 100% accuracy. Conversely, those skilled in the art should be aware of the possibility of an increase.

As used herein, "Possibility" and "Possibility" are measures of how likely it is to measure an event. It can be used interchangeably with "probability." Possibility refers to the probability of exceeding speculation but below certainty. Therefore, if a rational person uses common sense, training, or experience to arrive at a conclusion that the event is likely to be given in a given situation, then an event is possible. In some embodiments, a patient may or may not be treated with an M-CSF antagonist, eg, an M-CSF antibody (or continued treatment, or continued treatment in an increased dose) once the likelihood has been determined. (or discontinue treatment, or continue treatment with a reduced dose) patients.

The phrase "increased likelihood" refers to an increase in the probability that an event will occur. For example, some of the methods herein allow predictors whether patients will exhibit increased resistance to M-CSF compared to patients who do not have a specific CD163 performance level or who do not have an increased level of CD163 expression. The possibility of an anti-agent, for example, a M-CSF antibody treatment response or increased response to treatment with an M-CSF antagonist is preferred.

As used herein, the phrase "increased CD163 level" refers collectively to nucleic acid products, eg, an increase in RNA (eg, precursor-mRNA, mRNA, miRNA, etc.) and polypeptide products or fragments thereof.

"TAM-positive" refers to a CD163 level in a biopsy section that exceeds a predetermined threshold level that is predicted to be more likely to respond to an M-CSF antagonist as described herein. For example, a TAM positive patient can refer to a patient having an increase in CD163+ pixel density compared to a control group. In one embodiment, the predetermined threshold is set to capture about 40 to 50% of the TNBC sample with the highest TAM infiltration. In one embodiment, the TAM positive patient has more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 per biopsy slice when analyzed using quantitative IHC. , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50% of patients with CD163 pixel density (or TAM density).

The term "specific binding" as used in the context of a polypeptide means that the probe line binds to a given polypeptide target (eg, CD163), rather than a non-desired polypeptide that is randomly bound. However, "specific binding" does not exclude cross-reactivity with some undesired polypeptides, as long as the cross-reactivity does not interfere with the ability of the probe to provide a useful measure of the presence of a given polypeptide target.

The use of the term "capable" means the ability to achieve a given result, for example, the ability of a probe to detect the presence of a particular substance means that the probe can be used to detect a particular substance.

"Oligonucleotide" refers to a short nucleotide sequence, for example, 2 to 100 bases.

Various aspects of the invention are described in more detail in the following paragraphs and subsections.

Figure 1 is a graph showing the primary breast cancer samples stained with anti-CD163 antibody conjugated to DAB on two tissue microarrays, and the "TAM density".

Figure 2 is a graph showing predicted population mean serum concentration profiles of free M-CSF antibody H-RX1 after 10 mg/kg Q3W iv infusion with or without a loading dose.

The present invention relates to novel methods of treating patients suffering from breast cancer. In particular, the invention relates to the use of an M-CSF antagonist, such as an antibody described herein, for the treatment of breast cancer, such as triple-negative breast cancer. Currently, there is no target therapy for this breast cancer subtype and the only treatment option is chemotherapy. The present invention further provides a first personalized therapy that maximizes the benefit and minimizes the benefit of using an M-CSF antagonist in a breast cancer population prior to treatment with M-CSF therapy by identifying patients who are likely to respond favorably. The present invention encompasses the identification of patients with breast cancer, such as triple-negative breast cancer patients, having a cancer-associated macrophage (TAM) level indicative of a patient's likely response to treatment with an M-CSF antagonist. In particular, measuring a patient's TAM level and a patient's CD163 level by measuring the level of CD163 (eg, mRNA or protein) in a patient sample is then used to indicate whether the patient is more likely to respond favorably to M-CSF therapy. In one example, a patient is identified as a patient more likely to respond to an M-CSF antagonist if the patient has an increased CD163 level compared to a control group. In another example, if the patient has an amount of CD163 protein equal to or greater than the predetermined CD163 level ("cutoff"), then the patient is identified as a patient more likely to respond to the M-CSF antagonist. The level of CD163 expression analyzed in samples obtained from breast cancer patients can be, for example, mRNA expression and/or protein.

Preparation of samples

Any suitable sample or test sample taken from cells of an individual suffering from breast cancer can be used. In general, a test sample or tissue sample of a cell will be obtained from an individual suffering from cancer by biopsy or surgical resection. Samples of cells, tissues or fluids can be removed by needle aspiration biopsy. To this end, the fine needle attached to the syringe is inserted through the skin and inserted into the tissue of interest. Needles are usually guided to the area of concern using ultrasound or computed tomography (CT) imaging. Once the needle is inserted into the tissue, a vacuum is created with the syringe to make it thin The cells or fluid can be inhaled through the needle and collected in a syringe. Samples of cells or tissues can also be removed by incision or core biopsy. To this end, remove the cone, cylinder, or a small amount of tissue from the area of concern. CT imaging, ultrasound or endoscopy are commonly used to guide this type of biopsy. More specifically, the entire cancerous lesion can be removed by resection biopsy or surgical resection. In the present invention, the test sample is typically a sample of cells that have been partially removed by surgical resection.

For example, tissue test samples can also be stored, for example, in RNAlater (Ambion; Austin Tex.) or snap frozen and stored at -80 °C for later use. The sliced tissue sample can also be fixed with a fixative such as formaldehyde, paraformaldehyde, or acetic acid/ethanol. The fixed tissue sample can be immersed in wax (paraffin) or plastic resin. The soaked tissue sample (or frozen tissue sample) can be cut into thin sections. RNA or protein can also be extracted from fixed or wax-soaked tissue samples or frozen tissue samples. Once a cell sample or tissue sample has been removed from an individual suffering from cancer, it can be treated to isolate RNA or protein using techniques well known in the art and as described below.

An example of extracting RNA from a biopsy section taken from a patient suffering from cancer may include, for example, guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al, Biochemistry 18: 5294-5299, 1979) ). RNA from a single cell can be obtained as described in Methods for Preparing a cDNA Library from a Single Cell (see, for example, Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena et al, J. Immunol. Methods 190 :199, 1996). In one embodiment, the RNA population can be enriched for CD163. Linear amplification of in vitro transcription can be performed, for example, by random hexamer and primer-specific cDNA synthesis, or by multiple rounds of cDNA synthesis and template-directed (see, for example, Wang et al., Proc. Natl. Acad. Sci). .USA 86:9717, 1989; Dulac et al., supra; Jena et al., supra) to achieve enrichment.

An individual having a tumor or cancer will typically be a mammalian individual, such as a primate. In an exemplary embodiment, the individual is a human.

Analysis of TAM

The presence of TAM can be assessed by detecting the presence of CD 163.

The methods disclosed herein use, inter alia, the determination of the level of CD163. The level of CD163 predicts whether an individual is more likely to respond to an M-CSF antagonist. In one example, the predictive level of CD163 refers to a level of performance above the median level of CD163 expression in the breast cancer (control group) or greater than a predetermined cut-off value.

In one embodiment, the level of CD163 protein expression is compared to a control (or cut-off) to screen for treatment with a M-CSF antagonist, for example, a CD163 performance level compared to a control group predicts that the patient has It may or may not be possible to react with an M-CSF antagonist such as H-RX1. In one embodiment, a patient having a performance level (also referred to as "TAM density") with a CD163 protein performance exceeding a threshold level is selected for treatment with an M-CSF antagonist. For example, TAM% (pixel density = TAM density) can be determined by quantitative immunohistochemistry by measuring the % of CD163 positive pixels in a breast cancer biopsy section sample. In various embodiments, patients screened for treatment will exhibit performance levels that exceed the threshold CD163 performance level. In one embodiment, a patient having a performance level ("TAM density") with a CD163 protein performance exceeding a threshold level is selected for treatment with an M-CSF antagonist. The TAM density measurement of the biopsy section can show, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 35, 40, 45, 50% or higher CD163+ pixels, where the pixel density is equal to the TAM density. In various embodiments, the above density can be considered to be TAM positive and the patient will be selected for treatment by the methods described herein. The control level or threshold level of CD163 can be determined substantially simultaneously with the measurement of CD163 performance or can be determined in advance.

Detection of CD163 nucleic acid expression

The CD163 expression of a patient's biological sample, such as the presence of mRNA, can be analyzed by any suitable method. An increase in the level of CD163 expression can be used to predict an increase in the response to M-CSF antagonism in patients with breast cancer, for example, TBNC.

A number of methods and devices are available for determining the presence of CD163 nucleic acid expression. In some cases, the CD163 gene can be determined by measuring various levels of mRNA (or reverse transcribed cDNA) using various techniques, such as PCR-based analysis, reverse transcriptase PCR (RT-PCR) analysis, northern blotting, and the like. The level of performance. Quantitative RT-PCR can also be used.

In one example, the method comprises determining the performance of CD163 by using an agent that can be used for a particular detection gene, eg, RNA transcribed from a gene. The method can comprise providing a nucleic acid probe comprising a nucleotide sequence, for example, at least 10, 15, 25 or 40 nucleotides, and at most all or nearly all of the coding sequence partially complementary to one of the coding sequences of the nucleic acid sequence of CD163. Needle; obtain tissue samples from individuals with breast cancer; expose nucleic acid probes to RNA obtained from biopsy sections of patients taking breast cancer under stringent conditions (eg, in northern blots, in situ hybridization analysis, PCR) And etc.); and determining the amount of hybridization of the probe to the RNA. Nucleic acids can be labeled during or after enrichment and/or amplification of RNA. The biomarker CD163 is intended to also include naturally occurring sequences (including dual gene variants) and other family members. The CD163 biomarker of the present invention also includes a sequence which is complementary to the CD163 sequence due to degeneracy of the code, and a sequence which is sufficiently homologous and a sequence which hybridizes to CD163 under stringent conditions.

Conditions familiar to those skilled in the art for hybridization are found in Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred non-limiting example of one of the highly stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius followed by 0.2 X SSC, 0.1% SDS at 50 to 65 degrees Celsius. Or wash several times. By "sufficient homology" is meant a biomarker comprising a sufficient or minimum number of amino acids identical or equivalent to a second amino acid or nucleotide sequence (eg, an amino acid residue having a similar side chain). The residue or nucleotide is such that the first and second amino acid or nucleotide sequences share a common domain or motif and/or a functionally active amino acid or nucleotide sequence. For example, a common amino acid having a cross-domain amino acid sequence of at least about 50% homology, at least about 60% homology, at least about 70%, at least about Amino acid or nucleotide sequences of 80%, and at least about 90 to 95% homology are defined herein as sufficiently homologous. Additionally, at least about 50% homology, at least about 60 to 70% homology, at least about 70 to 80%, at least about 80 to 90%, and at least about 90 to 95% of the amine groups sharing a common functional activity An acid or nucleotide sequence is defined herein as sufficiently homologous.

Mathematical algorithms can be used to achieve comparisons between sequences and the determination of percent homology between two sequences. A preferred non-limiting example of one of the mathematical algorithms for comparison of sequences is Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68 as in Karlin and Altschul (1993) Proc. The algorithm modified in Natl.Acad.Sci.USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. MoI. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequence homology to the TRL nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequence homology to the CD163 protein sequence. To achieve gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Research 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the preset parameters of the respective programs (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov for details. Another preferred non-limiting example of a mathematical algorithm for comparison of sequences is the ALIGN algorithm of Myers and Miller, CABIOS (1989). When using the ALIGN program to compare amino acid sequences, a PAM1 20 weight residue table, a 12 gap length penalty, and a 4 gap penalty can be used.

The invention includes measuring the performance of CD163 in a tumor biopsy section from an individual suffering from breast cancer. The level of performance can be analyzed and used to generate a score that can be used to distinguish between patients with tumors that are likely to respond to M-CSF and those who do not respond to M-CSF. In one example, the level of performance of CD163 is measured and analyzed and used to generate a performance threshold that can be used to screen for individuals that are more likely to respond to M-CSF antagonists. In a In one embodiment, an individual having a higher level of CD163 expression than the control, for example, at least 10%, 15%, 20%, 30%, 40% or greater is selected.

It may be necessary to correct (normalize) both the difference in the amount of RNA analyzed and the variability in the quality of the RNA used. Therefore, analysis typically measures and incorporates the performance of certain standardized genes or housekeeping genes.

CD163 expression can be determined using any method known in the art, such as reverse transcriptase PCR (RT-PCR). The method involves the use of any technique known in the related art, for example, by detaching mRNA by using a purification kit, a buffer setting, and a protease from a manufacturer such as Qiagen. The reverse transcription step is typically initiated using a specific primer, random hexamer or oligo-dT primer, and the derived cDNA can then be used as a template in subsequent PCR reactions depending on the nature of the performance and the target. TaqMan(R) RT-PCR can then be performed using, for example, commercially available equipment.

An up-to-date change in RT-PCR technology is real-time quantitative PCR, which measures the accumulation of PCR products by dual-labeled fluorescent generation probes (eg, using TaqMan(R) probes). The real-time PCR is compatible not only with the quantitative competitive PCR used by the internal competitors of each target sequence for standardization, but also with the standardized gene contained in the sample or the housekeeping gene for RT-PCR. Rong. For further details see, for example, Held et al, Genome Research 6: 986-994 (1996).

As used herein, a control group for comparison can be determined by those skilled in the art. In one aspect, the control group is determined by selecting the value to be used as the cutoff value. For example, the value can be a value that distinguishes, for example, a test sample with an increased CD163 performance from a display exhibiting the lowest or presence of a CD163 performer.

Detection of CD163 protein

In some cases, the presence of CD163 can be determined by analyzing the CD163 polypeptide product. Any known method in the related art may be used, including but not limited to, immunocytochemical staining, ELISA, flow cytometry, Western blotting, spectrophotometry, HPLC and mass spectrometry were used to detect the polypeptide product.

One method for detecting a polypeptide product in a sample is by a probe that is a binding protein that is capable of specifically interacting with a labeled protein (eg, an antibody capable of binding to a CD163 protein). Preferably, a labeled antibody, a binding moiety thereof, or other binding partner can be used. The antibody may be of single or multiple strains or may be made biosynthetically. The binding partner can also be a naturally occurring molecule or synthetically produced. The amount of the composite protein is determined using standard protein detection methods described in the related art. Detailed reviews, theories, and protocols for immunological analysis design can be found in many textbooks in the related art, including Practical Immunology, Butt, W. R., Marcel Dekker, New York, 1984. Various assays can be used to detect proteins with labeled antibodies. Direct labels include fluorescent or luminescent labels attached to antibodies, metals, dyes, radionuclides, and the like. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, catalase, and the like. In a one-step assay, the polypeptide product, if present, is fixed and cultured with the labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove unbound molecules, the label of the sample is analyzed.

The invention also contemplates the use of immobilized antibodies specific for a protein or polypeptide. The antibody can be immobilized to a variety of solid supports, such as magnetic or chromatography matrix particles, surfaces of assays (such as microtiter wells), solid substrate material sheets (such as plastic, nylon, paper), and the like. Analytical bands can be prepared by coating the antibody or a plurality of antibodies on a solid support in an array. The strip can then be immersed in the test sample and then quickly processed by the wash and detection steps to produce a detectable message, such as a stain.

In a one-step analysis, the immobilized polypeptide product of CD163 or CD163 protein can be cultured with an unlabeled antibody. The unlabeled antibody complex, if present, binds to a second labeled antibody specific for the unlabeled antibody. Wash the sample and analyze the presence of the label. The choice of marker for labeling antibodies will vary depending on the application. However, the choice of marking can be easily determined by those skilled in the art. The antibody can be labeled with a radioactive atom, an enzyme, a chromogenic activity or a fluorescent moiety, or a colorimetric label. The choice of marker mark will also change depending on the desired detection limit. Enzyme assays (ELISA) typically allow the detection of colored products formed by the interaction of an enzyme-labeled complex with an enzyme substrate. Some examples of radioactive atoms include ³² P, ¹²⁵ I, ³ H, and ¹⁴ P. Some examples of enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Some examples of chromogenically active moieties include luciferin and rhodamine. Antibodies can be coupled to such labels by methods known in the art. For example, the enzyme and chromogenic active molecules can be coupled to the antibody by a coupling agent such as dialdehyde, carbodiimide, bismaleimide, and the like. Alternatively, the coupling can occur via a ligand-receptor pair. Some suitable ligand-receptor pairs include, for example, biotin-avidin or streptavidin, and antibody-antigen.

In one aspect, the invention encompasses the use of a sandwich technique for detecting a polypeptide product in a biological sample. This technique requires two antibodies that bind to the protein of interest: for example, an antibody is immobilized on a solid support and an antibody is free in solution, but is labeled with some readily detectable chemical compound. Examples of chemical labels that can be used for the second antibody include, but are not limited to, radioisotopes, fluorescent compounds, and enzymes or other molecules that produce colored or electrochemically active products upon exposure to a reactant or enzyme substrate. When a sample containing the polypeptide product is placed in the system, the polypeptide product binds to both the immobilized antibody and the labeled antibody. The result is a "sandwich" immune complex on the surface of the support. The composite protein is detected by washing to remove unbound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed with the protein on the surface of the support. Sandwich immunoassays are highly specific and extremely sensitive if markers with good detection limits are used.

Western blot analysis is well known to those skilled in the art (Sambrook et al, Molecular Cloning, A Laboratory Manual, 1989, Vol. 3, Chapter 18, Cold Spring Harbor Laboratory). In Western blots, samples were separated by SDS-PAGE. Transfer the gel to the membrane. The membrane is incubated with labeled antibodies to detect the desired protein.

In another embodiment, IHC is used to detect and quantify CD163 protein in tumor biopsy sections as a measure of TAM density in a tumor. In one example, a mouse monoclonal anti-human CD163 antibody (pure line MRQ-26; Ventana Medical Systems, Tuscon, AZ, USA) can be used on formalin-immobilized paraffin-fixed paraffin on an automated Ventana immunohistochemical staining instrument. Immunostaining of CD163 was performed on tissue sections of soaked (FFPE) tumor material. Tumor morphology and its relationship to CD163 staining specific for TAM can then be assessed by pathologists on digitally scanned slides to define and define relevant regions of interest (ROI) in the tumor. The area of CD163 staining in the ROI can then be analyzed by automated imaging analysis algorithms and platforms (for a review, see references Hamilton et al. 2014, Methods 70 (2014) 59-73, Mullane et al. 2008, Expert Rev Mol Diagn, 2008 8 (6) 707-725) Quantification to determine the percentage of tumor area occupied by TAM as a measure of "TAM density" in the tumor.

In the methods of the invention described herein, patients are screened for treatment based on the level of performance of CD163. In one example, the patient is selected when the patient has a particular level of protein performance compared to a suitable control sample.

Control group

As appropriate herein, the control group for comparison can be determined by those skilled in the art. In one example, a control group is determined by selecting a control sample having a TAM density value that defines a cutoff value that is more likely to respond to an M-CSF antagonist and a less likely responder. In one embodiment, the threshold is determined to identify the TNBC patient with the highest TAM infiltration (this is expected to represent approximately 40% of the TNBC patient). The predicted individual has equal to or greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 as measured by quantitative IHC Test samples of CD163 levels (or TAM densities) at 27, 28, 29, 30, 35, 40, 45, 50% CD163 pixel density are more likely to respond to M-CSF antagonists. In certain embodiments, it is unlikely that a patient is predicted to have a cutoff or threshold of, for example, 8% CD163 pixel density or less by quantitative IHC measurements. The response to treatment and therefore not selected for treatment. In another example, the control group can be a sample from a healthy volunteer or a sample from a breast cancer patient known to have low CD163 performance (mRNA or protein), and when the measured CD163 value is greater than CD163 in such a control group. The patient is selected for treatment.

In yet another example, the control value may be a value based on a mathematical model predetermined by the prior control group. The model (also known as a classifier) can be established by using a level of performance (eg, mRNA or protein) of a batch of breast cancer patients (eg, TBNC). The mathematical model can be used as a threshold for, for example, any type of prediction method or its variations and derived control values. If the performance level is above or above the threshold, the patient is more likely to respond to treatment with an M-CSF antagonist. In one embodiment, individuals having a CD163 performance level equal to or greater than the threshold, for example, may be 8%, 10%, 15%, 20%, 30%, 40% or higher.

data analysis

Information regarding the presence of CD 163 may be stored in any of the methods described herein for determining the presence of CD 163, for example, the presence of CD 163 protein. Typically, a physician or genetic counselor or patient or other researcher's results can be informed once the CD163 performance level or CD163 protein level is determined. In particular, the results can be delivered in a form that can be transmitted or transmitted to other researchers or physicians or genetic counselors or patients. This form may vary and may be physical or non-physical. The results in the individual being tested may be embodied in an illustrative statement, schema, photograph, chart, image or any other visual form. For example, statements regarding CD163 nucleic acid expression levels or CD163 protein levels can be used to indicate test results. Such statements and visual forms may be recorded on physical media (such as paper, computer readable media (such as floppy disks), optical disks, etc.), or non-physical media (for example, in the form of a website on an email or the Internet or an intranet) On the electronic media). In addition, the results can also be recorded in sound form and transmitted via any suitable medium, such as analog or digital cable, fiber optic cable, etc., by telephone, fax, wireless mobile, internet telephony, and the like. All such forms (both physical and non-entity) will constitute "transportable form of information" formula". Therefore, information and data about test results can be generated and transmitted to a different location anywhere in the world. For example, when an analysis is conducted outside the country, information and data about the test results can be generated and delivered in a transmittable form as described above. Test results in a transferable form can therefore be entered into the United States. Accordingly, the present invention also encompasses a method for producing a deliverable form comprising information on CD163 nucleic acid expression levels or CD163 protein levels. This form of information can be used to predict the responsiveness of a patient with breast cancer to treatment with an M-CSF antagonist alone or in combination with another agent, such as a chemotherapeutic agent, to select a treatment based on this information, and to selectively treat based on this information. patient.

Screening and treatment of patients with breast cancer

CD163 nucleic acid expression or CD163 protein levels allow physicians to provide a personalized therapy for breast cancer patients, such as TBNC, that is, it allows for the determination of whether a patient is selectively treated with an M-CSF antagonist. In this way, physicians can maximize benefits and minimize the risk of M-CSF antagonism in a population of patients suffering from breast cancer.

Macrophage Community Stimulating Factor (M-CSF), also known as Community Stimulating Factor (CSF-1)

The full length human M-CSF mRNA line encodes 554 amino acid precursor proteins. In alternative mRNA ligation and differential translation post-translational proteolysis, M-CSF may be a transmembrane sugar on the surface of cells containing proteoglycan glycoproteins or chondroitin sulfate secreted into the circulation or expressed as cells producing M-CSF. protein. The three-dimensional structure of the 150 amino acids at the amino terminus of human M-CSF (the smallest sequence required for full in vitro biological activity) indicates that the protein is composed of four alpha helix bundles and antiparallel beta The disulfide-bonded dimer consists of a sheet (Pandit et al., Science 258: 1358-62 (1992)). Three different M-CSF species are produced by alternative mRNA ligation. The three polypeptide precursors are M-CFS alpha of 256 amino acids, M-CSF beta of 554 amino acids, and M-CSFγ of 438 amino acids. M-CSFβ is a secreted protein that is not present in a membrane-bound form. M-CSFα appears as an intact membrane protein that is slowly released by decomposition of the cleavage of the protein. M-CSFα is cleaved at the amino acid 191-197 of the sequence set forth in SEQ ID NO 11. Membrane-bound form of M-CSF can be associated with nearby cells Receptor interactions and thus mediate specific intercellular contacts. The term "M-CSF" may also include the amino acid 36-438 of SEQ ID NO: 13.

M-CSF binds to various forms of the function of the receptor M-CSFR on its target cells. M-CSFR is a transmembrane molecule with five extracellular immunoglobulin-like domains, a transmembrane domain, and an intracellular disrupted Src-associated tyrosine kinase domain. M-CSFR is encoded by the c-fms proto-oncogene. Binding of M-CSF to the extracellular domain of M-CSFR results in receptor dimerization, which activates the cytoplasmic kinase domain, resulting in autophosphorylation and phosphorylation of other cellular proteins (Hamilton JA, J Leukoc Biol., 62(2): 145-55 (1997); Hamilton J, A., Immuno Today., 18(7): 313-7 (1997).

SEQ ID NO: 14 is the amino acid sequence of M-CSFα. SEQ ID NO: 15 is the amino acid sequence of M-CSFβ. SEQ ID NO: 16 is the amino acid sequence of M-CSFγ. Several polymorphisms in the DNA sequence can result in differences in amino acids. For example, the common polymorphism provides Ala instead of Pro at location 104.

Examples of M-CSF antagonists

M-CSF antagonists are known in the related art. The M-CSF antagonist can be a small molecule, antibody or other antigen binding protein, a small molecule, a nucleic acid (such as an siRNA), or any other such molecule that interferes with M-CSF activation or function.

In one example, M-CSF antagonists suitable for use in the methods of the presently disclosed methods include antibodies or fragments thereof.

The M-CSF antibody which can be used in the present invention includes the M-CSF antibody which is disclosed in International Publication No. WO 2005/068503, which is incorporated by reference in its entirety for its teachings on M-CSF antibodies. Incorporated herein. WO 2005/068503 discloses (e.g.) with the antibody and RX1,5H4, MC1 and / or antigenic determinants of an antibody binding the same bit MC3, pharmaceutical formulations comprising an antibody of the M-CSF-specific antibody of Human Engineered ^TM type, and the preparation of Method of pharmaceutical formulation. The term "antibody" is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments that bind to an antigen (eg, Fab', F) '(ab)2, Fv, single-chain antibody, diabody), and the recombinant peptide described above, as long as it exhibits a desired biological activity.

Other M-CSF antibodies that can be used in the present invention include M-CSF antibodies disclosed in International Publication Nos. WO 2003/028752, US2009117103, and US2005059113, each of which is based on its teachings on M-CSF antibodies. It is incorporated herein by reference in its entirety.

In one embodiment the method of the present invention, the antibody is a Human Engineered ^TM RX1 antibody or antigen binding fragment thereof. The following is a table showing the heavy and light variable regions and CDRs of RX1, and their CDRs.

In one embodiment, an antibody or fragment thereof suitable for use in the methods of the invention comprises an antibody or fragment thereof that binds to a linear epitope as represented by RFRDNTPN (SEQ ID NO: 11) or RFRDNTAN (SEQ ID NO: 12). Such an antibody is an RX1 antibody. In another embodiment, the antibody can be an antibody or fragment thereof that binds to a linear epitope determined by ITFEFVDQE (SEQ ID NO: 13). Such an antibody is a 5H4 antibody.

In another embodiment suitable for use in the methods of the invention, the antibody is a human engineered RX1 antibody ("H-RX1" as used herein) or an antigen-binding fragment thereof. The following is a show H- A table of the weight and light variable regions and CDRs of RX1.

In one embodiment, the antibody is a humanized antibody or human engineered antibody or fragment thereof comprising the heavy chain variable region sequence set forth in SEQ ID NO: 1 and the light chain variable region sequence set forth in SEQ ID NO: .

In yet another embodiment, the antibody or fragment thereof comprises a heavy chain variable region comprising a CDR1, CDR2 and CDR3 domain; and a light chain variable region comprising a CDR1, CDR2 and CDR3 domain, wherein the heavy chain variable region The light chain variable region CDR3 comprises an amino acid having the sequence set forth in SEQ ID NO: 7; and the light chain variable region CDR3 comprises an amino acid having the sequence set forth in SEQ ID NO: 10; and the antibody or Its antigen-binding portion binds to human M-CSF with a binding affinity of about 10 ^-7 M. The antibody or fragment thereof may further comprise a heavy chain variable region CDR2 comprising an amino acid having the sequence set forth in SEQ ID No: 6; and a light chain comprising an amino acid having the sequence set forth in SEQ ID NO: Variable region CDR2. The antibody or fragment thereof may further comprise a heavy chain variable region CDR1 comprising an amino acid having the sequence set forth in SEQ ID NO: 5; and a light chain comprising an amino acid having the sequence set forth in SEQ ID NO: Variable region CDR1.

In yet another example, a humanized antibody or human engineered antibody or fragment thereof suitable for use in the methods of the invention binds to human M-CSF, wherein the anti-system and M-CSF comprise RFRDNTPN (SEQ ID NO: 11) Or the epitope binding of at least 4 adjacent residues of RFRDNTAN (SEQ ID NO: 12), wherein the antibody has an affinity Kd (dissociation equilibrium constant) of at least 10 ^-7 M against human M-CSF, wherein the antibody comprises SEQ ID NO: 5, all three heavy chain CDRs of SEQ ID NO: 6 and SEQ ID NO: 7 and all three light chain CDRs of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

The antibodies disclosed herein may be derivatives of single chain antibodies, bivalent antibodies, domain antibodies, nanobodies, and unibodies. For example, the invention provides an isolated monoclonal antibody (or a functional fragment thereof) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino group relative to SEQ ID NO: An amino acid sequence having at least 80%, at least 90%, or at least 95% identity of the acid sequence; the light chain variable region comprising at least 80%, at least 90%, or at least relative to the amino acid sequence of SEQ ID NO: A 95% consensus amino acid sequence; the anti-system binds to M-CSF (eg, human and/or cynomolgus M-CSF) and neutralizes the signaling activity of M-CSF. Also included within the scope of the invention are variable heavy and light chain parent nucleotide sequences; and full length heavy and light chain sequences that are optimized for expression in mammalian cells. Other antibodies of the invention include amino acids or nucleic acids that have been mutated but have at least 60%, 70%, 80%, 90%, 95% or 98% identity to the above sequences. In some embodiments, which include a variable region in which the variable region is described in comparison to the sequence described above No more than 1, 2, 3, 4 or 5 amino acids have been deleted, inserted or replaced by a mutant acid amino acid sequence by amino acid.

In other embodiments, the variable heavy (VH) and/or variable light (VL) amino acid sequences can have 50%, 60%, 70%, 80%, 90 relative to the sequences set forth in Table 1 above. %, 95%, 96%, 97%, 98% or 99% consistency. In other embodiments, the VH and/or VL amino acid sequences may be the same except for no more than 1, 2, 3, 4 or 5 amino acid positions in the amino acid positions. The VH and VL regions having antibodies relative to those described in Table 1 can be obtained by mutation (e.g., site-directed mutagenesis or PCR-mediated mutation) followed by functional assays described herein to test the function of encoding the altered antibody. High (i.e., 80% or greater) consistent antibody to the VH and VL regions.

In other embodiments, the variable region of the heavy and/or light chain nucleotide sequence can be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% consistency.

And X. Specific amino acid sequences based on the antibodies described herein or their conserved modifications, and the desired functional properties of such antibodies retaining the M-CSF binding antibodies described in Table 1.

Accordingly, the present invention provides an isolated M-CSF monoclonal antibody or fragment thereof, which comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences Wherein: the heavy chain variable region CDR1 amino acid sequence is selected from the group consisting of SEQ ID NO: 5 and its conserved modifications; the heavy chain variable region CDR2 amino acid sequence is selected from the group consisting of SEQ ID NO: a group of 6 and a conserved modification thereof; the heavy chain variable region CDR3 amino acid sequence is selected from the group consisting of SEQ ID NO: 7 and its conserved modifications; the light chain variable region CDR1 amino acid sequence Selected from the group consisting of SEQ ID NO: 8 and its conserved modification; the light chain variable region CDR2 amino acid sequence is selected from SEQ ID NO: 9 and a population thereof comprising a conserved modification; the light chain variable regions of the CDR3 amino acid sequence are selected from the group consisting of SEQ ID NO: 10 and its conserved modifications; the antibody or fragment thereof is specifically Bind to M-CSF and neutralized M-CSF activity.

The antibody used in the present invention may be a fragment of an antibody that binds to M-CSF selected from the group consisting of: Fab, F(ab ₂ )', F(ab) ₂ ', scFv, VHH, VH, VL, dAbs .

The invention also encompasses antibodies which interact with the same epitope as the M-CSF binding antibody described above (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution).

The antibodies of the invention may comprise various antibody isoforms or mixtures thereof, such as IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Typically, they include IgG1 (eg, IgG1k) and IgM isoforms. Human sequence antibodies can be full length (eg, IgGl or IgG4 antibodies) or can comprise only antigen binding portions (eg, Fab, F(ab') ₂ , Fv or single chain Fv fragments).

In one embodiment, the invention encompasses a light chain comprising a heavy chain variable region sequence as set forth in SEQ ID NO: 4 and an IgGl constant region and a light chain variable region sequence comprising the sequence of SEQ ID NO: A humanized antibody or fragment thereof. The light chain can further comprise a human kappa constant region. In a preferred embodiment, the present invention provides a modification of Human IgG1 or IgG4 constant region Engineered ^TM variant comprises an antibody or a modified or not. In the case of IgGl, modifications of the constant region, in particular the hinge or CH2 region, may enhance or attenuate effector function, including ADCC and/or CDC activity. In other embodiments, the IgG2 constant region is modified to reduce antibody-antigen aggregate formation. In the case of IgG4, modification of the constant region, specifically the hinge region, can reduce the formation of half antibodies. In a specific exemplary embodiment, a mutation of the IgG4 hinge sequence Cys-Pro-Ser-Cys (SEQ ID NO: 17) to the IgGl hinge sequence Cys-Pro-Pro-Cys (SEQ ID NO: 18) is provided. ^(TM) has been shown to contain an antibody IgG1 or Engineered Human IgG4 constant region of the antibody having improved properties compared to Human Engineered ^TM comprising the constant region of IgG2 (WO 2005/068503). Selection of the IgGl or IgG4 Fc region improves binding affinity, M-CSF neutralizing activity, and anti-osteoclast activity. Furthermore, the selection of the IgGl or IgG4 Fc region provides antigen-antibody complexes that are more similar to those of the parental murine antibody. The mobility in the hinge region therefore appears to significantly affect the binding of the antibody to the dimeric antigen MCSF as well as the antibody neutralizing activity. The invention generally encompasses the preparation of antibodies comprising a modified or unmodified IgGl or IgG4 constant region, in particular a hinge and a CH2 domain, or preferably at least a heavy chain of the hinge domain, enhancing binding affinity and/or subtraction The slow antibody dissociates from the dimeric antigen.

Additional antibody sequences encompassed by the present invention are provided in WO 2005/068503, as described herein. The specific sequences disclosed in WO 2005/068503 and intended for use in the compositions and methods described herein are as follows.

Additional antibody sequences encompassed by the present invention are provided in WO 2005/068503, as described herein. The specific sequences disclosed in WO 2005/068503 and intended for use in the compositions and methods described herein are as follows:

Combination of antibodies

In another aspect, the invention relates to an M-CSF antagonist (eg, an antibody) or a fragment thereof in combination with other therapeutic agents for treating cancer. In particular, M-CSF antagonists can be combined with cancer chemotherapeutic agents or radiation therapy or surgery. Cancer chemotherapeutic agents include, but are not limited to, alkylating agents such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents such as carmustine (BCNU); Antimetabolites, such as methotrexate; anthraquinone analogs, antimetabolites, mercaptopurine; pyrimidine analogs, anti-metabolites, such as fluorouracil (5-FU) and gemcitabine; hormone antineoplastic agents, such as goserelin (goserelin), leuprolide and tamoxifen; natural anti-tumor agents such as aldesleukin, interleukin-2, docetaxel, etopo Etoposide (VP-16), interferon alpha, paclitaxel and retinoic acid (ATRA); natural anti-tumor agents for antibiotics, such as bleomycin, dactinomycin , daunorubicin, doxorubicin and mitomycin; and vinca alkaloids natural anti-tumor agents, such as vinblastine, vincristine, vindesine (vindesine); hydroxyurea; aceglatone, doxorubicin (adri) Amycin), ifosfamide, enocitabine, epitiostolol, aclarubicin, ancitabine, nimustine, Procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3), anti-tumor polysaccharide, anti-tumor platelet factor, cyclophosphamide, Schizophyllan, cytarabine, dacarbazine, thioinosine, thiophene Thietepa, tegafur, neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan ( Busulfan), honvan, peplomycin, bestatin (ubenimex), interferon-β, mepitiostane, dibromomannitol ), merphalan, laminin peptide, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen) /methylchlorethamine).

In one example, the M-CSF antagonists shown in Table 1 above, for example, M-CSF antibodies, such as the H-RX1 line, are administered with the chemotherapeutic agent carboplatin. In another example, the M-CSF antagonist is administered with the chemotherapeutic agent gemcitabine. In yet another embodiment, the M-CSF antagonists shown in Table 1 above, for example, M-CSF antibodies, such as the H-RX1 line, are administered with the chemotherapeutic agents carboplatin and gemcitabine.

Carboplatin is an alkylating agent that acts by cross-linking guanine bases in the DNA double helix strand, thereby preventing transcription and replication. The mechanism of action is not cell cycle specific (McEvoy, GK, American Hospital Formulary Service Drug Information, American Society of Health System (2000)). The main route to remove carboplatin is renal excretion. Patients with normal renal function had a 71% excretion dose within 24 hours. Drug interactions with aglycosides, phenytoin and warfarin have been reported and combined for attention (McEvoy 2000). The main toxicity of carboplatin is myelosuppression, nausea/vomiting, neurotoxicity and peripheral neuropathy. Allergic reactions have been reported, especially when used in combination or after repeated exposures (McEvoy 2000, Markman, M. et al., J. Clin. Oncol., 17(4): 1141 (1999)). Carboplatin has been approved for use in ovarian and non-small cell lung cancer alone or in combination with other drugs, but is also widely used Other malignant diseases, such as head and neck cancer, germ cell tumors, breast cancer and others. There are many dosing regimens that vary depending on the disease, response expectations, and combination therapy (NCCN 2014, ESMO Guidelines 2014).

Gemcitabine is a pyrimidine analog that is metabolized into two active metabolites, gemcitabine diphosphate and gemcitabine triphosphate. The cytotoxic effect of gemcitabine is achieved by incorporation of gemcitabine triphosphate into DNA, resulting in inhibition of DNA synthesis and apoptosis. Gemcitabine is cell cycle-specific (S and G1/S-phase) (Grindey, G. et al., Cancer Invest., 8(2): 313 (1990)). Gemcitabine is rapidly metabolized by cytosine deaminase in liver, kidney, blood and other tissues (Gilbert, J. et al, Clin. Cancer. Res., 12: 1794-1803 (2006)). Drug elimination is mainly mediated through renal excretion. Within one week of administration, 92% to 98% of the dose is almost completely recovered in the urine. The main toxicities of gemcitabine are elevated liver enzymes [aspartate transaminase (ASAT)/aspartate aminotransferase (ALAT)] and alkaline phosphatase (ALK), nausea/vomiting, protein/hematuria, Difficulty breathing, rash and myelosuppression. Gemcitabine has been approved for use in pancreatic cancer, ovarian cancer, non-small cell lung cancer, and breast cancer, either alone or in combination with other drugs. The most commonly used regimen for gemcitabine is administered on Days 1 and 8 of each 21-day cycle, but there are several options (NCCN 2014, ESMO Guidelines 2014).

Pharmaceutical composition and investment

A pharmaceutical or sterile composition comprising an M-CSF antagonist in admixture with a pharmaceutically acceptable carrier or excipient is prepared. The compositions may additionally comprise one or more additional therapeutic agents, such as the chemotherapeutic agents described above.

Formulations of therapeutic and diagnostic agents can be prepared by mixing with a physiologically acceptable carrier, excipient or stabilizer, for example, in the form of a lyophilized powder, slurry, aqueous solution, emulsion or suspension (see, For example, Hardman et al, (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (ed.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

The choice of treatment regimen for treatment depends on several factors, including the rate of serum or tissue renewal of the entity, the extent of the symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with the extent of acceptable side effects. Thus, the amount of biodelivery depends in part on the particular entity and the severity of the condition being treated. There are currently guidelines for selecting suitable doses of antibodies, interleukins, and small molecules (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al., (2003) New Engl. J. Med. 348:601 - 608; Milgrom et al, (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al, (2001) New Engl. J. Med. 344: 783-792; Beniaminovitz et al, (2000) New Engl. J. Med. 342: 613-619; Ghosh et al., (2003) New Engl. J. Med. 348: 24-32; Lipsky et al., (2000) New Engl. J. Med. 343: 1594 -1602).

Determination of the appropriate dosage is made by the physician, for example, using parameters or factors known or suspected in the relevant art that affect the treatment or prediction that would affect the treatment. In general, the dose begins with an amount that is slightly less than the optimal dose and thereafter increases in a small increase until relatively any side effect. Achieve the desired or best effect. Important diagnostic measures include, for example, the level of inflammation or the level of inflammatory interleukin produced.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to achieve an amount of the active ingredient which is effective to achieve a therapeutic response to a particular condition, composition, and mode of administration, and which is non-toxic to the patient. The selected dosage level will vary depending on a variety of factors that may affect the pharmacokinetics, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of treatment of the particular compound employed, the duration of treatment, Other drugs, compounds and/or materials used in combination with the particular compositions used, age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors known in medical technology .

Compositions comprising an antibody or fragment thereof of the invention may be provided by continuous infusion or by administration, for example, at intervals of one day, one week, or one to seven times per week. The dose can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracranically, or by inhalation. A particular dosage regimen is for the maximum dose or dose frequency that avoids many undesirable side effects. Each given dose may be at least 0.05 μg/kg, at least 0.2 μg/kg, at least 0.5 μg/kg, at least 1 μg/kg, at least 10 μg/kg, at least 100 μg/kg, at least 0.2 mg/kg, at least 0.3. Mg/kg, at least 0.5 mg/kg, at least 1.0 mg/kg, at least 2.0 mg/kg, at least 3.0 mg/kg, at least 5.0 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25mg/kg or at least 50mg/kg body weight (or other body type descriptions, eg, ideal body weight (IBW), body mass index (BMI), body surface area (BSA), fat free mass (FFM), lean body mass (LBW), adjustment Body weight (ABW), ideal body weight percentage (%IBW), and predicted normal body weight (PNWT)) (see, for example, Yang et al., (2003) New Engl. J. Med. 349:427-434; Herold et al., ( 2002) New Engl. J. Med. 346: 1692-1698; Liu et al, (1999) J. Neurol. Neurosurg. Psych. 67: 451-456; Portielji et al, (2003) Cancer Immunol. Immunother. 52: 133-144, Green & Duffull, (2004) Br J Clin Pharmacol 58:2 119-133.). The desired dose of the antibody or fragment thereof is about the same as the antibody or polypeptide, in moles per kg body weight. The desired serum or plasma concentration of the antibody or fragment thereof is about the same as the antibody or polypeptide, in moles per kg body weight. The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg or at least 100 μg. The dosage administered to the individual can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or greater.

For the antibody or fragment thereof of the invention, the dosage administered to the patient can range from 0.01 mg/kg to 100 mg/kg of patient body weight (or other body type description). The dose may be between 0.01 mg/kg and 20 mg/kg, 0.01 mg/kg and 10 mg/kg, 0.01 mg/kg and 5 mg/kg, 0.01 and 3 mg/kg, 0.01 and 2 mg/kg, 0.01 and 1 mg/kg, 0.01 mg/kg and 0.75 mg/kg, 0.01 mg/kg and 0.5 mg/kg, 0.01 mg/kg to 0.25 mg/kg, 0.01 to 0.15 mg/kg, or 0.01 to 0.10 mg/kg of patient body weight (or other body type) Description) between. In one example, the dosage may be based on body weight, for example, 0.01 mg/kg, 0.03 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg, 30 mg/kg, 40 mg/kg. 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg. The dose can be delivered in a stationary or fixed amount, for example, 0.75 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 75 mg, 150 mg, 300 mg, 500 mg, 700 mg, 1000 mg or 1500 mg.

The unit dose of the antibody or fragment thereof of the present invention may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5. Mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg , 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg To 2.5mg.

The dose of the antibody or fragment thereof of the invention may be at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml in the individual. At least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml At least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, at least 400 μg/ml, at least 425 μg/ml, at least 450 μg/ml A serum titer (or serum or plasma concentration) of at least 475 [mu]g/ml, or at least 500 [mu]g/ml. Alternatively, the dose of the antibody or fragment thereof of the invention may be at least 0.1 μg/ml, at least 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 10 μg/ in the individual. Ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200 μg/ml, at least 225 μg/ Serum titers of ml, at least 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml.

The dose of the antibody or fragment thereof of the present invention can be repeated and administered at least 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 30 days, 42 days. , 45 days, 56 days, 2 months, 75 days, 3 months, or at least 6 months.

The effective amount of a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the methodological route, and the severity of the dosage and side effects (see, for example, Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

In one embodiment, the M-CSF anti-system is administered to the patient at a dose of between about 0.1 and 20 mg/kg at time 0 ("first administration"). Can then be used as needed after the first investment An additional dose ("extra dose" or "loading dose") is administered from day 7 to day 14. The M-CSF antibody was then administered every three weeks after the first administration. In another embodiment, the M-CSF antibody, eg, the H-RX1 line, is administered to the patient at a dose of about 10 mg/kg at time 0 ("first administration"). An additional dose of 10 mg/kg ("Additional Dose") was then administered on the 8th day after the first administration. The M-CSF antibody was then administered every three weeks after the first administration. The basic principle of adding an additional dose is to reach a steady state earlier, thereby achieving a faster and more continuous inhibition of M-CSF.

The route of administration may be by, for example, topical or dermal administration, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracranial, intralesional injection or infusion, or by sustained release system or implantation. Inclusions (see, for example, Sidman et al., (1983) Biopolymers 22: 547-556; Langer et al., (1981) J. Biomed. Mater. Res. 15: 167-277; Langer (1982) Chem. Tech. 12: 98-105; Epstein et al, (1985) Proc. Natl. Acad. Sci. USA 82: 3688-3692; Hwang et al, (1980) Proc. Natl. Acad. Sci. USA 77: 4030-4034; U.S. Patent Nos. 6,350,466 and 6,316,024). If desired, the composition may also contain a solubilizing agent and a local anesthetic (e.g., lidocaine to reduce pain at the injection site). In addition, pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and with a nebulizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication No. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety.

In one embodiment, the M-CSF anti-system is between time 0.1 ("first dose") between about 0.1 and 20 mg/kg, for example, 0.1, 0.3, 1, 3, 5, 6, 7, 8 The dose of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg is administered intravenously to the patient, which can then be administered as needed on the 7th day after the first administration. 14 days, 7 days, On the 8th, 9th, 10th, 11th, and 12th days, an additional dose ("extra dose") was administered and then M-CSF antibody was administered every three weeks after the first administration. In another embodiment, the M-CSF anti-system is administered to the patient at a dose of about 10 mg/kg at time 0 ("first administration"). An additional dose of about 10 mg/kg ("extra dose") was then administered on the 8th day after the first administration. Then about 10 mg/kg of M-CSF antibody was administered every three weeks after the first administration. The basic principle of adding an additional dose is to achieve a steady state earlier, thereby achieving a faster and more continuous inhibition of M-CSF.

The compositions of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. Parenteral routes of administration can be used, for example, by injection or infusion. Parenteral administration may refer to modes of administration other than enteral and topical administration (usually by injection) and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of the invention may be administered by a non-injectable route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, an antibody or fragment thereof of the invention is administered by infusion. In another embodiment, the multispecific epitope binding protein of the invention is administered subcutaneously.

If an antibody or fragment thereof of the invention is administered in a controlled release or sustained release system, a pump can be used to achieve controlled or sustained release (see, Langer, supra; Sefton, (1987) CRC Crit. Ref Biomed. Eng .14:20; Buchwald et al. (1980), Surgery 88: 507; Saudek et al., (1989) N. Engl. J. Med. 321: 574). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (ed.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al, (1985) Science 228: 190; Although et al, (1989) Ann. Neurol. 25: 351; Howard et al, (1989) J. Neurosurg. 7 1:105); U.S. Patent No. 5, 679, 377; U.S. Patent No. 5,916, 597; U.S. Patent No. 5,912, 015; U.S. Patent No. 5, 989, 463; U.S. Patent No. 5,128, 326; PCT Publication No. WO 99/15154; And PCT Publication No. WO 99/20253. Examples of polymers for use in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-copolymer) -vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol) ), polylactide (PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoester. In one embodiment, the polymer used in the sustained release formulation is inert, free of filterable impurities, storage stable, sterile, and biodegradable. The control or sustained release system can be placed adjacent to a prophylactic or therapeutic target, thus requiring only one part of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 ( 1984)).

If the antibody or fragment thereof of the present invention is administered topically, it can be formulated into an ointment, a cream, a transdermal patch, an emulsion, a gel, a shampoo, a spray, an aerosol, a solution, an emulsion, Or other forms that are well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th Edition, Mack Pub. Co., Easton, Pa. (1995). For topical dosage forms which are not sprayable, it is customary to use a carrier comprising one or more excipients which are compatible with topical application and which have a viscous to semi-solid or solid form with a dynamic viscosity (in some cases greater than water). Suitable formulations include, but are not limited to, solutions, suspensions, lotions, creams, ointments, powders, liniments, ointments and the like, which are sterilized or auxiliary if necessary (eg, preservatives, stabilizers) The wetting agent, buffer or salt is mixed to affect various properties such as, for example, osmotic pressure. Other suitable topical dosage forms include Sprayable aerosol formulation wherein, in some cases, the active ingredient in combination with a solid or liquid inert carrier is packaged or packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant such as a freon). Squeeze the bottle. If desired, moisturizers can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art.

If the compositions comprising the antibodies or fragments thereof are administered intranasally, they may be formulated in the form of an aerosol, spray, mist, or drops. In particular, prophylactic or therapeutic agents for use in the present invention may conveniently be in the form of an aerosol spray from a pressurized pack or nebulizer by using a suitable propellant (eg, dichlorodifluoromethane, trichloro Delivery of fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. Capsules and mashes (consisting of, for example, gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

Methods suitable for co-administration or treatment with a second therapeutic agent are known in the art (see, for example, Hardman et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition Supplement, McGraw -Hill, New York, NY; Poole and Peterson (ed.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (ed.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of the therapeutic agent can reduce symptoms by at least 10%; at least 20% less; at least about 30%; at least 40%, or at least 50%.

In one embodiment, the M-CSF is administered with another agent such as a chemotherapeutic agent such as gemcitabine, carboplatin, cisplatin, and the like.

M-CSF antagonists and other inhibitors or agents can be administered periodically. Periodic therapy involves administering a first therapy (eg, a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second therapy (eg, a second prophylactic or therapeutic agent) for a period of time, as appropriate, Then administering a third therapy (eg, a prophylactic or therapeutic agent) for a period of time, etc., and repeating the continuous administration (ie, cycle) to reduce the development of resistance to one of the therapies to avoid Or reduce the side effects of one of these therapies, and / or improve the efficacy of the therapy.

In certain embodiments, an antibody or fragment thereof of the invention can be formulated to ensure proper in vivo distribution. For example, the blood brain barrier (BBB) rejects many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB, if desired, they can be formulated, for example, as liposomes. For a method of making a liposome, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Such liposomes may comprise one or more moieties that are selectively transported into a particular cell or organ thereby enhancing targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). . Exemplary target moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); Mannose (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038) Antibody (Bloeman et al, (1995) FEBS Lett. 357: 140; Owais et al, (1995) Antimicrob. Agents Chemother. 39: 180); Surfactant Protein A Receptor (Briscoe et al, (1995) Am .J. Physiol.1233: 134); p. 120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123 JJKillion; IJ Fidler (1994) Immunomethods 4:273.

The invention provides for the administration of an M-CSF antagonist, alone or in combination with one other therapy. The combination therapy of the present invention (e.g., a prophylactic or therapeutic agent) can be administered to an individual simultaneously or sequentially. Therapies of the combination therapies of the invention may also be administered periodically. Periodic therapy involves administering a first therapy (eg, a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second therapy (eg, a second prophylactic or therapeutic agent) for a period of time and repeating the continuous administration (ie, cycle) to reduce the development of resistance to one of the therapies (eg, agents) to avoid or reduce one of the therapies (eg, agents) Side effects of the therapy, and / or improve the efficacy of the therapy.

Combination therapy with an M-CSF antagonist and other agents can be administered to an individual simultaneously. The term "simultaneously" is not limited to administration of therapy (eg, a prophylactic or therapeutic agent) at exactly the same time, and conversely, it means that the pharmaceutical composition comprising the antibody or fragment thereof of the invention is in order and The individual is administered over a time interval such that the antibodies of the invention can work in conjunction with other therapies to provide a higher benefit than if they were otherwise administered. For example, each therapy may be administered to the individual sequentially in any order at the same time or at different time points; however, if not administered at the same time, their administration time should be close enough to provide the desired therapeutic or Preventive effects. Each therapy can be administered to the individual separately in any suitable form and in any suitable route. In various embodiments, the therapy (eg, prophylactic or therapeutic agent) is less than 15 minutes, less than 30 minutes, less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, at intervals From about 2 hours to about 3 hours, at intervals of from about 3 hours to about 4 hours, at intervals of from about 4 hours to about 5 hours, at intervals of from about 5 hours to about 6 hours, at intervals of from about 6 hours to about 7 hours, at intervals of about 7 hours to about 8 hours, interval of about 8 hours to about 9 hours, interval of about 9 hours to about 10 hours, interval of about 10 hours to about 11 hours, interval of about 11 hours to about 12 hours, interval of 24 hours, interval of 48 hours, interval of 72 hours Or at an interval of 1 week. In other embodiments, two or more therapies (eg, prophylactic or therapeutic agents) are administered at the same patient visit.

Combination therapies can be administered to an individual with the same pharmaceutical composition. Alternatively, the combination therapy can be administered to an individual simultaneously with a separate pharmaceutical composition. The therapeutic agent can be administered to the individual by the same or different routes of administration.

Set

The invention also encompasses kits for detecting CD163 expression or CD163 protein in a patient biological sample (test sample) and optionally a control sample. Such kits can be used to predict whether a patient with breast cancer is likely to respond (or have an enhanced response) to treatment with an M-CSF antagonist alone or in combination with another agent, such as a chemotherapeutic agent. For example, the kit can include A probe capable of detecting a CD163 expression level, a CD163 protein level or a CD163 activity level in a biological sample, a product of an equivalent gene signature of the allele and/or its allele (eg, an oligonucleotide, an antibody, Labeled compound or other agent). The kit may also include instructions for providing a prediction that the patient will be able to respond to treatment with an M-CSF antagonist alone or in combination with another agent.

The disclosed kits can also include, for example, buffers, preservatives, or protein stabilizers. The kit may also include components (eg, enzymes or substrates) required to detect the detectable agent. The kit can also contain a control sample or a series of control samples that can be analyzed and compared to the included test samples. The components of the kit are typically sealed in individual containers, and all of the various containers and instructions for use are in a single package for use.

Such kits may also include M-CSF antagonists, for example, M-CSF antibodies, such as H-RX1 (eg, in liquid or lyophilized form) or pharmaceutical compositions comprising M-CSF antagonists (as described above), And optionally, one or more additional therapeutic agents described herein (eg, a second agent (such as carboplatin) and a third agent (such as gemcitabine)) or a pharmaceutical composition comprising the agents. In this manner, the kits are suitable for the selective treatment of breast cancer patients using M-CSF antagonists. Additionally, the kits can include components for administering an M-CSF antagonist (eg, syringes and vials, prefilled syringes, prefilled pens) and instructions for use. Such kits can accommodate, for example, additional therapeutic agents for treating breast cancer (as described above) for delivery in combination with the encapsulated M-CSF antagonist.

The present invention has been fully described, and the invention is described by the following examples and claims, which are intended to be illustrative and not restrictive.

Instance

Example 1

CMCS110Z2201 is H-RX1 in a patient with advanced triple-negative breast cancer (TNBC) (having a heavy chain variable region comprising an amino acid as described in SEQ ID NO: 2 and comprising an amino acid as described in SEQ ID NO: 4) Light chain variable region antibody) in combination with carboplatin / gemcitab Bin (carboplatin/gem) for phase II, open labeling, randomization studies of individual carbo/gem. There was no substantial progression in TNBC treatment and the prognosis was still poor. Currently, chemotherapy is the only treatment option. Although TNBC has an excellent prognosis for a subset of chemosensitive diseases and chemotherapy responders, most patients relapse rapidly. Current TNBC treatment strategies aim to further improve the efficacy of chemotherapy. The target study population will include patients whose tumors contain high TAM immunoinvasion ("high TAM"). In this study, only patients with a measured 8% TAM content or higher as measured by detecting CD163 would be included.

● Select patients by detecting CD163 to detect TAM

Recently, gene expression patterns were used to describe six different subtypes of TNBC: two basal cells, one stromal cell, one mesenchymal stem cell, one luminal androgen receptor (LAR), and one immunomodulatory ( IM) subtype (Lehmann 2011, J Clin Invest. 121(7): 2750-67). Ongoing and emerging treatments include targeted therapies selected for each unique subtype. The IM subtype accounts for approximately 25% of all TNBCs and is characterized by an increase in the expression of genes involved in immune cell signaling such as T-cell function, immune transcription and interferon response (Lehmann 2014). In addition, the IM subtype exhibits substantially higher levels of tumor infiltrating lymphocytes (TIL) and tumor-associated macrophages (TAM) than other TNBC subtypes (Mahmoud 2012, J Clin Pathol 65: 159-163 and Vinayak ASCO 2014, ASCO Annual Meeting J Clin Oncol 32:5s). Forty percent of TNBC has high TAM tumor infiltration (high TAM). The high TAM subgroup is rich in IM, capturing 75% of all IM (Yuan 2014, OncoTargets and Therapy 7: 1475-1480; Lehmann 2011, supra, Jaeger [TCGA] 2014, Novartis's own data analyzing the TCGA database).

M2 macrophages or TAM have been defined as CD163+, CD68+, but immunofluorescence staining of breast cancer tissue sections has confirmed that CD163 staining is consistent with CD68 staining. Immunohistochemical staining also confirmed co-staining of CD163 and CD68, however, CD163-DAB-conjugated antibodies produced a clearer background subtraction from the background of the CD68-DAB conjugate-conjugated antibody minus the image. image. Based on these data, single immunohistochemical staining with anti-CD163-DAB conjugated antibodies was chosen to detect M2 macrophages or TAM.

Two different tissue microarray containing breast cancer tumor samples were stained with anti-CD163-DAB conjugated antibody to detect TAM. IHC was performed using a Ventana Discovery XT automatic dyeing machine. Sections were deparaffinized, treated with Ventana Cell Regulatory #1 (CCIS) antigen extraction agent and then incubated with primary antibody (CD163 drug line ready-up) for 32 minutes at room temperature. Detection was performed using a ChromoMap kit (Roche/Ventana, cat# 760-159). All stained slides were digitized using an Aperio Scanscope full field scanner. The stained tumor sections of each sample were analyzed using an automated image analysis algorithm to determine the percentage of positive pixels in the total pixels covered by CD163-DAB staining, defined as a measure of the "TAM density". The TNBC sample shows the widest range of CD163 performance or "TAM density". For TNBC samples, a range of "TAM density" of ~25% to <1% is obtained. Based on the understanding that 75% of all IM subtypes of TNBC patients with the highest TAM infiltration capture TNBC, set a threshold (or lower limit), and define a high TAM population of ~12 to 15% TAM density, thereby capturing 40 to 50% of the highest TAM infiltrated TNBC sample.

Figure 1 shows a primary breast cancer sample on two tissue microarrays independently stained with anti-CD163 antibody conjugated to DAB, and "TAM density" was determined as described. Column 1 depicts TAM density values obtained for non-TNBC breast cancer samples and those who are HER2+/ER+/PR+; column 2 shows TAM density values obtained for TNBC samples. All samples in columns 1 and 2 were obtained from the University Hospital Basel tissue microarray, which was obtained from patients with various stages of the disease, including metastatic disease. All samples from column 3 and column 4 were obtained from Uppsala University, which were obtained from patients with non-advanced disease (T1-2/N0/M0). Column 3 depicts TAM density values obtained for non-TNBC breast cancer samples and those of HER2+/ER+/PR+; column 4 shows TAM density values obtained for TNBC samples. The data shows that the Microarray at the University of Basel Hospital identifies more samples with higher TAM densities, The array contains a larger number of advanced TNBC patient samples consistent. The images of the right markers A, B, C, D depict the actual CD163 stained tissue sections of the indicated patient samples; the TAM density values (in %) of each sample based on CD163 staining are indicated in the inset.

Based on CD163 staining, TAM infiltration was greatly increased in triple negative breast cancer (TNBC) compared to hormone receptor positive breast cancer samples. These findings show that CD163 staining in TNBC patients is a suitable marker for measuring changes in the CD163-positive TAM population following M-CSF signaling using M-CSF inhibitors.

Example 2

TNBC patients with TAM density based on CD163 staining received the M-CSF antagonist H-RX1 at 10 mg/kg iv every three weeks. A 3-week cycle was chosen to match the dosing cycle of chemotherapy (carbo/gem). In addition, a certain additional dose or loading dose of M-CSF was administered on the 8th day after the first administration.

The population PK model based on the development of self-healthy volunteers predicted a certain loading dose on day 8 of cycle 1, and the H-RX1 concentration reached steady state at week 6 instead of week 12. Achieving a faster TAM elimination and thus improved chemotherapeutic efficacy can be clinically important for TNBC in a given invasive clinical course. Figure 2 shows a graph of predicted population mean serum concentration profiles of free H-RX1 after 10 mg/kg Q3W iv infusion with and without an additional dose.

The basic principle of adding an additional dose is to achieve a steady state earlier, thus achieving a faster and more continuous inhibition of H-RX1. The population PK model based on the development of self-healthy volunteer data was predicted to utilize an additional dose on day 8 of cycle 1, and the H-RX1 concentration reached steady state at week 6 instead of week 12. Achieving a faster TAM elimination and thus improved chemotherapeutic efficacy can be clinically important for TNBC in a given invasive clinical course.

Example 3

A 78-year-old woman diagnosed with metastatic TNBC was found to have a high TAM content (more than 10% TAM) in her tumor biopsy sections and was therefore eligible for inclusion in the study. The patient No adjuvant or neoadjuvant therapy for breast cancer has been previously received; the disease has metastasized at the time of diagnosis. The patients were randomized to receive treatment with the H-RX1 combination carboplatin/gemcitabine. After 2 treatment cycles, the tumor status was assessed and the patient's tumor index lesion was found to have a 40% reduction (ie, partial response to treatment). Especially for the patient, the target 1 (left breast tumor) was reduced from 52.9 x 34.8 mm to 47.1 x 32.8 mm. Target 2 (right axillary lymph node) was reduced from 16.4 mm to 5 mm. Target 3 (right axillary lymph node) was reduced from 18.6 to 5.3 mm. Target 4 (left inguinal lymph node) was reduced from 26 mm to 14 mm. Target 5 (paraluminal lymph nodes) was reduced from 16.2 mm to 7.9 mm. Treatment with carboplatin and gemcitabine alone resulted in an objective response rate of approximately 30%. The results of the present invention indicate that combination therapy with H-RX1 can lead to further benefits.

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Claims (22)

A method of treating a patient suffering from triple-negative breast cancer comprising administering to the patient a therapeutically effective amount of an M-CSF antibody or fragment thereof, wherein the M-CSF antibody or fragment comprises 1, 2, 3, 4, Five or six CDRs as shown in Table 1. A method of treating a patient suffering from breast cancer comprising selectively administering to the patient a therapeutically effective amount of an M-CSF antibody or fragment thereof based on the patient having a level of CD163 expression predicting that it is likely to respond to the M-CSF antibody or fragment thereof And wherein the antibody or fragment comprises 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. A method of selectively treating a patient suffering from breast cancer, comprising: a) screening a patient for treatment with a therapeutically effective amount of an M-CSF antagonist based on the patient having a likelihood that the patient is predicted compared to a control group The level of CD163 expression in response to M-CSF; and b) thereafter, a therapeutically effective amount of an M-CSF antagonist is administered. A method of selectively treating a patient suffering from breast cancer, comprising: a) analyzing an elevated level of CD163 performance in a patient biological sample; and b) thereafter, screening a patient for treatment with a therapeutically effective amount of an M-CSF antagonist, It is based on the fact that the patient's biological sample has a level of CD163 expression predicting that the patient is likely to respond to M-CSF compared to the control group; and c) thereafter, the M-CSF antagonist is administered to the screened patient. A therapeutically effective amount of an M-CSF antagonist suitable for treating a patient suffering from breast cancer, wherein: a) screening for a patient treated with an M-CSF antagonist based on the patient having a predicted ratio compared to a control group The patient may have a CD163 performance level for M-CSF response; b) Thereafter, a therapeutically effective amount of an M-CSF antagonist is administered. A method for predicting the likelihood that a patient suffering from breast cancer will be treated with a therapeutically effective amount of an M-CSF antagonist, comprising analyzing the level of CD163 performance of the patient's biological sample, wherein: a) the level of CD163 expression is compared to the control group. There is an increase in the likelihood that the patient will increase the response to treatment with the M-CSF antagonist; and b) there is no increase in the level of CD163 expression compared to the control, indicating that the patient will respond to treatment with the M-CSF antagonist. The possibility is reduced. A method of selectively treating a breast cancer patient comprising: a) analyzing a level of a CD163 protein of a patient's biological sample; and b) thereafter, screening a patient for treatment with an M-CSF antagonist based on the patient's biological The sample has a level of CD163 expression that predicts that the patient is likely to respond to M-CSF compared to the control group; and c) thereafter, the M-CSF antagonist is administered to the screened patient. The method of any one of claims 6 to 7, wherein the expression of the CD 163 is analyzed using quantitative immunohistochemistry. The method of any one of claims 1 to 8, wherein the administering step comprises administering to the patient a dose of between about 5 and 20 mg/kg of M-CSF antagonist at time 0 ("first administration "), a dose ("extra dose") was administered again on the 7th to 14th day after the first administration, and then every 3 weeks after the first administration. The method of any of the above claims, wherein the M-CSF antagonist is an M-CSF binding molecule. The method of claim 10, wherein the M-CSF binding molecule is an M-CSF antibody or a fragment thereof. The method of claim 11, wherein the antibody or fragment thereof comprises 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. The method of claim 11, wherein the isolated M-CSF antibody or fragment thereof comprises: VH comprising SEQ ID NO: 2 and VL comprising SEQ ID NO: 4, or 90%, 91%, 92% thereof , 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the amino acid sequence. A method of making information in a transmissible form of predicting the reactivity of a patient suffering from breast cancer to treatment with an M-CSF antagonist, comprising: a) analyzing the level of CD163 performance of a patient's biological sample; and b) applying to the dissemination The results of the analysis step are recorded in physical or non-physical media form, wherein if the result is that the CD163 performance level is higher than the control group, it indicates that the patient will have an increased likelihood of responding to the treatment comprising the M-CSF antagonist. A method of treating a patient suffering from breast cancer comprising: a) administering a combination of: i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent (simultaneously, separately or sequentially) Sexually), and iii) a therapeutically effective amount of a third agent (simultaneously, separately or sequentially). A method of selectively treating a patient suffering from breast cancer, comprising: a) analyzing an elevated level of CD163 protein in a patient's biological sample; b) screening for a therapeutically effective amount of an M-CSF antagonist, ii) treatment An effective amount of a second agent, and iii) a combination of a therapeutically effective amount of a third agent, based on the patient having a CD163 performance level predicting that the patient is likely to respond to M-CSF compared to a control group; c) Thereafter, the following combination is administered simultaneously, separately or sequentially: i) a therapeutically effective amount of an M-CSF antagonist, ii) a therapeutically effective amount of a second agent, and iii) a therapeutically effective amount The third agent. The method of any one of claims 15 to 16, wherein the breast cancer is TNBC. The method of claim 16, wherein the level of CD163 analyzed is protein. The method of any one of clauses 15 to 18, wherein the M-CSF antagonist is M-CSF Antibody or fragment thereof. The method of claim 19, wherein the antibody or fragment thereof comprises 1, 2, 3, 4, 5 or 6 CDRs as shown in Table 1. The method of any one of claims 15 to 20, wherein the second agent is carboplatin and the third agent is gemcitabine. A method of selectively treating a patient suffering from TNBC comprising: simultaneously, separately or sequentially administering to the patient the following combination: i) a therapeutically effective amount comprising one, two, three, 4, 5 or 6 M-CSF antibodies or fragments thereof, such as the CDRs shown in Table 1, ii) a therapeutically effective amount of carboplatin, and iii) a therapeutically effective amount of gemcitabine based on the patient's prediction This patient is more likely to have a CD163 level of response to treatment with this combination.