CN114901308A - Compositions and methods for treating cancer using anti-HER 2 antibody drug conjugates - Google Patents

Compositions and methods for treating cancer using anti-HER 2 antibody drug conjugates Download PDF

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CN114901308A
CN114901308A CN202080076131.1A CN202080076131A CN114901308A CN 114901308 A CN114901308 A CN 114901308A CN 202080076131 A CN202080076131 A CN 202080076131A CN 114901308 A CN114901308 A CN 114901308A
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antibody
cancer
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dp303c
amino acid
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冯立新
丽莎·艾伦
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Jushi Biopharmaceutical Co ltd
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Abstract

The present application provides compositions and methods for treating cancer using an antibody-drug conjugate (ADC) comprising an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugate moiety is conjugated to the acceptor glutamine residue.

Description

Compositions and methods for treating cancer using anti-HER 2 antibody drug conjugates
Cross Reference to Related Applications
This application claims priority to U.S. provisional application No. 62/927,623 filed on 29/10/2019, the contents of which are hereby incorporated by reference in their entirety for all purposes.
ASCII text file submission sequence listing
The following is submitted in an ASCII text file and is incorporated herein by reference in its entirety: computer Readable Form (CRF) of sequence Listing (filename: 720692000240SEQLIST. TXT, date recorded: 10 months and 19 days 2020, size: 14 KB).
Technical Field
The present application is in the field of cancer therapeutics and relates to compositions and methods for treating cancer using antibody-drug conjugates (ADCs).
Background
The human epidermal growth factor receptor (HER) family plays an important role in the pathogenesis of many tumors. The HER2 receptor is overexpressed in many tumors (25% breast, 20% ovarian, 30% intestinal gastric, 20% lung) and this overexpression is associated with aggressive tumors and poor prognosis (Slamon DJ et al, Science, 1987, 235(4785): 177; Slamon DJ et al, Science, 1989, 244(495): 707; Morrison C et al, J. Clin. Oncol., 2006, 24(15): 2376). Despite recent advances in the development of therapeutic agents targeting HER2, patients with metastatic HER 2-positive breast and gastric cancers are rarely cured of disease. Prototype antibody-drug conjugates (ADC) against HER2, Enmetuzumab (Ado-Trastuzumab emtansine, KADCYLA), have been approved for the treatment of HER2 overexpressing cancer. However, its use is limited to breast cancer.
The FDA approved us prescription information for KADCYLA @ includes data from a phase 3 trial in women with advanced HER 2-positive breast cancer who have received prior therapy with taxane and trastuzumab. The enzmetuzumab group exhibited statistically significant improvements in objective response rate (43.6%), progression-free survival (median 9.6 months), and overall survival (median 30.9 months) compared to the control group treated with lapatinib and capecitabine. Prescription information includes framed warnings of hepatotoxicity, cardiotoxicity and embryofetal toxicity. Additional warnings and precautions are listed for pulmonary toxicity, infusion-related reactions, bleeding, thrombocytopenia, and neurotoxicity. As shown in the society discussing the results from more recent experiments with Enmetuzumab (K. Jhaveri, J Clin Oncol. 2017; 35(2): 127-.
There remains a need for improved therapeutic agents to treat patients with HER 2-positive cancer. A novel ADC that improves the efficacy and/or safety profile of emmetruzumab may provide a beneficial therapeutic alternative for patients with HER 2-positive cancer.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.
Brief description of the invention
In one aspect, the invention provides a method of treating HER 2-positive cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, and wherein the conjugate moiety is conjugated to the receptor glutamine residue. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an Immunohistochemistry (IHC) test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a Fluorescence In Situ Hybridization (FISH) test.
In some embodiments according to any of the above methods, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the individual has previously received a second HER2-targeting agent. In some embodiments, the HER 2-positive cancer is resistant or refractory to a second HER2-targeting agent. In some embodiments, the second HER-2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib.
In some embodiments according to any of the above methods, the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1mg/kg to about 2 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 3.0 mg/kg.
In one aspect, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1mg/kg to about 2 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2mg/kg to about 3 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 2.0 mg/kg. In some embodiments, the antibody-drug conjugate is administered at a dose of about 3.0 mg/kg.
In some embodiments according to any of the above methods, the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once a week. In some embodiments, the individual is a human.
In some embodiments according to any of the above methods, the cancer is a solid cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer. In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the subject has failed a previous cancer therapy, such as an anti-Her 2 antibody therapy (e.g., trastuzumab).
In some embodiments according to any of the above methods, the Fc region of the anti-HER 2 antibody is N-glycosylated. In some embodiments, the receptor glutamine residue is Q295 in the heavy chain of an anti-HER 2 antibody according to EU numbering.
In some embodiments according to any one of the above methods, each heavy chain of the HER2 antibody is conjugated to a conjugate moiety. In some embodiments, the conjugate moiety is conjugated to the acceptor glutamine residue by transglutaminase amidation.
In some embodiments according to any of the above methods, the anti-HER 2 antibody comprises: a heavy chain variable region (VH) comprising: heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and a light chain variable region (VL) comprising: light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the anti-HER 2 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO. 7 and a VL comprising the amino acid sequence of SEQ ID NO. 8. In some embodiments, the Fc region is IgGl Fc. In some embodiments, the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10.
In some embodiments according to any of the above methods, the conjugate moiety comprises a cleavable linker. In some embodiments, the toxin is monomethyl auristatin e (mmae). In some embodiments, the conjugate moiety has the chemical structure of formula (III):
Figure 506802DEST_PATH_IMAGE001
wherein n is an integer selected from 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (I):
Figure 627205DEST_PATH_IMAGE002
in some embodiments, the conjugate moiety is LND 1002.
In some embodiments according to any of the above methods, the antibody-drug conjugate is DP303 c.
Kits and articles of manufacture for use in any of the above methods are also provided.
It is to be understood that one, some or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art.
Brief Description of Drawings
Fig. 1 depicts a schematic structure of DP 001.
FIG. 2 shows cell proliferation inhibition curves of SK-BR-3 cells (HER 23 + cell line) after treatment with DP001 (blue circles and blue lines), DP303c (green triangles and green lines) or T-DM1 (brown squares and brown lines).
FIG. 3 shows cell proliferation inhibition curves of JIMT-1 cells (HER 22 + cell line) after treatment with DP001 (blue circles and blue lines), DP303c (green triangles and green lines), or T-DM1 (brown squares and brown lines).
FIG. 4 shows inhibition curves of cell proliferation of Hs746T cells (HER2 negative cell line) after treatment with DP001 (blue circles and blue lines), DP303c (green triangles and green lines), or T-DM1 (brown squares and brown lines).
FIG. 5 shows the effect of DP303c on the growth of the human gastric cancer xenograft model NCI-N87. Athymic nude mice were implanted with NCI-N87 cells (HER 23 + cell line). Blank controls (PBS, gray filled circles and gray lines), DP303c at 2mg/kg (purple open diamonds and purple dotted lines), DP303c at 4 mg/kg (purple closed diamonds and purple dashed lines), DP303c at 8 mg/kg (purple open diamonds and purple solid lines), T-DM1 at 2mg/kg (black open squares and black dotted lines), T-DM1 at 4 mg/kg (black closed squares and black dashed lines), or T-DM1 at 8 mg/kg (black closed squares and black solid lines) administered intravenously to the animals in single doses. Tumor size was measured at the indicated time points. Data for each treatment group are presented as mean values.
FIG. 6 shows the effect of DP303c on the growth of JIMT-1, a human breast cancer xenograft model. Athymic nude mice were implanted with JIMT-1 cells. The animals were administered a single dose intravenously with a placebo (control), DP303c, T-DM1, or BP-ADC. Tumor size was measured at the indicated time points. There were 5 animals in each group. Data for each treatment group are presented as mean values.
FIG. 7 shows the serum DP303c concentration-time curves after a single IV bolus of 3 mg/kg (blue diamonds and blue long dashes), 10 mg/kg (red squares and red dashes) or 30mg/kg (grey triangles and grey solid lines) DP303c in SD rats. Data represent mean ± standard deviation.
FIG. 8 shows the serum total antibody (DP001) concentration versus time curve after a single IV bolus of 3 mg/kg (blue diamonds and blue long dashes), 10 mg/kg (red squares and red short dashes) or 30mg/kg (green triangles and green solid lines) DP303c in SD rats. Data represent mean ± standard deviation.
FIG. 9 shows the free MMAE concentration-time curves after a single IV bolus of 3 mg/kg (blue diamonds and blue long dashes), 10 mg/kg (red squares and red dashes) or 30mg/kg (green triangles and green solid lines) DP303c in SD rats. Data represent mean ± standard deviation.
FIG. 10 shows the serum concentration-time curve for DP303c after a single intravenous infusion of 1.2 mg/kg (squares and dotted lines), 4 mg/kg (triangles and dashed lines), or 12mg/kg (diamonds and solid lines) DP303c in cynomolgus monkeys. Data represent mean ± standard deviation.
FIG. 11 shows the serum concentration-time curve for total antibody (DP001) after a single intravenous infusion of 1.2 mg/kg (squares and dotted lines), 4 mg/kg (triangles and dashed lines), or 12mg/kg (diamonds and solid lines) DP303c in cynomolgus monkeys. Data represent mean ± standard deviation.
FIG. 12 shows plasma concentration-time curves of free MMAE after a single intravenous infusion of 1.2 mg/kg (squares and dotted lines), 4 mg/kg (triangles and dashed lines), or 12mg/kg (diamonds and solid lines) DP303c in cynomolgus monkeys. Data represent mean ± standard deviation.
Figure 13 shows plasma concentration-time curves of free MMAE after two intravenous infusions of 6 mg/kg (blue squares and dotted line) or 20mg/kg (red triangles and dashed line) DP303c in cynomolgus monkeys. Data represent mean ± standard deviation.
Figures 14A and 14B show serum concentration-time curves of DP303c after intravenous infusion of 0, 6, 20/12, 40/30mg/kg DP303c in cynomolgus monkeys. The serum DP303c concentration-time curves show the following groups and infusions: group 2 after the first dose (6.0 mg/kg, day 1, open circle), group 3 after the first dose (20.0 mg/kg, day 1, open square), group 4 after the first dose (40.0 mg/kg, day 1, closed square), group 2 after the fourth dose (6.0 mg/kg, day 64, open triangle), group 3 after the fourth dose (12.0 mg/kg, day 64, closed triangle), group 4 after the third dose (30.0 mg/kg, day 43, closed circle). Figure 14A shows the serum concentration-time curve of DP303c in male cynomolgus monkeys. Figure 14B shows the serum concentration-time curve of DP303c in female cynomolgus monkeys.
Figures 15A and 15B show serum concentration-time curves of total antibody (DP001) after intravenous infusion of 0, 6, 20/12, 40/30mg/kg DP303c in cynomolgus monkeys. The serum total antibody (DP001) concentration-time curves show the following for each group and infusion: group 2 after the first dose (6.0 mg/kg, day 1, open circle), group 3 after the first dose (20.0 mg/kg, day 1, open square), group 4 after the first dose (40.0 mg/kg, day 1, closed square), group 2 after the fourth dose (6.0 mg/kg, day 64, open triangle), group 3 after the fourth dose (12.0 mg/kg, day 64, closed triangle), group 4 after the third dose (30.0 mg/kg, day 43, closed circle). Figure 15A shows the serum concentration-time curve of total antibody (DP001) in male cynomolgus monkeys. Figure 15B shows the serum concentration-time curve of total antibody (DP001) in female cynomolgus monkeys.
Figures 16A and 16B show serum concentration-time curves of free MMAE after intravenous infusion of 0, 6, 20/12, 40/30mg/kg DP303c in cynomolgus monkeys. The serum free MMAE concentration-time curves show the following for each group and infusion: group 2 after the first dose (6.0 mg/kg, day 1, open circles), group 3 after the first dose (20.0 mg/kg, day 1, open squares), group 4 after the first dose (40.0 mg/kg, day 1, open triangles), group 2 after the fourth dose (6.0 mg/kg, day 64, closed circles), group 3 after the fourth dose (12.0 mg/kg, day 64, closed squares), group 4 after the third dose (30.0 mg/kg, day 43, open triangles). Figure 16A shows serum concentration-time curves of free MMAE in male cynomolgus monkeys. Figure 16B shows serum concentration-time curves of free MMAE in female cynomolgus monkeys.
Detailed Description
The present application provides methods of treating HER 2-positive cancer in an individual using antibody-drug conjugates (ADCs) whose toxin components are conjugated to endogenous receptor glutamine residues in the glycosylated Fc region of an anti-HER 2 antibody. In some embodiments, the ADC is DP303 c. The ADCs described herein have improved conjugation stability in vitro and in vivo, which helps to increase efficacy against HER 2-positive cancers (such as HER 22 + and 3+ cancers) and reduce adverse reactions. The methods described herein are useful for treating various HER 2-positive solid cancers, including those that are resistant to standard HER2-targeted therapies.
Accordingly, in one aspect, the application provides a method of treating HER 2-expressing cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, and wherein the conjugate moiety is conjugated to the receptor glutamine residue. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the subject is resistant or refractory to a second HER2-targeting agent, such as trastuzumab, enritumumab, pertuzumab, or lapatinib. In some embodiments, the antibody-drug conjugate is DP303 c.
In another aspect, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to 3 mg/kg). In some embodiments, the antibody-drug conjugate is administered intravenously. In some embodiments, the antibody-drug conjugate is administered once every three weeks, once every other week, or once a week. In some embodiments, the antibody-drug conjugate is DP303 c.
I. Definition of
As used herein, "HER 2" refers to human epidermal growth factor receptor 2. "HER 2-positive cancer" refers to a cancer that overexpresses HER2 on cancer cells as compared to noncancerous normal cells. HER2 status can be determined using known HER2 tests including Immunohistochemistry (IHC) tests, Fluorescence In Situ Hybridization (FISH) tests, subtractive probe technique chromogenic in situ hybridization (SPoT-Light HER2 CISH) tests and Inform double in situ hybridization (Inform HER2 Dual ISH) tests. HER 2-positive cancers include cancers that test as 2+ (borderline) or 3+ (positive) in an IHC test. HER 2-positive cancers also include cancers that test positive in the HER2 FISH test, the SPoT-Light HER2 CISH test, and/or the Inform HER2 Dual ISH test. "HER 22 + cancer" refers to a cancer that tests as 2+ in an IHC assay. "HER 23 + cancer" refers to a cancer that tests as 3+ in the IHC test.
As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of a disease, stabilizing a disease (e.g., preventing or delaying the worsening of a disease), preventing or delaying the spread of a disease (e.g., metastasis), preventing or delaying the recurrence of a disease, reducing the rate of recurrence of a disease, delaying or slowing the progression of a disease, improving the state of a disease, providing remission (partial or total) of a disease, reducing the dose of one or more other drugs required to treat a disease, delaying the progression of a disease, improving the quality of life, and/or prolonging survival. In some embodiments, the treatment reduces the severity of one or more symptoms associated with the cancer by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to the corresponding symptom in the same subject prior to treatment or as compared to the corresponding symptom in other subjects not receiving the treatment. "treatment" also encompasses a reduction in the pathological consequences of cancer. The methods of the invention contemplate any one or more of these therapeutic aspects.
The terms "recurrence (recurrence)", "recurrence (relapse)" or "recurrence (relapsed)" refer to the re-appearance of cancer or disease after clinical assessment of disease remission. Diagnosis of distant metastasis or local recurrence may be considered recurrence.
The term "refractory" or "resistant" refers to a cancer or disease that is not responsive to treatment.
An "adjuvant setting" refers to a clinical setting in which an individual has a history of cancer and is generally, but not necessarily, responsive to therapy, including but not limited to surgery (e.g., surgical resection), radiation therapy, and chemotherapy. However, due to their history of cancer, these individuals are considered to be at risk of developing the disease. Treatment or administration in the "adjuvant setting" refers to the subsequent mode of treatment. The degree of risk (e.g., when an individual in the adjuvant setting is considered "high risk" or "low risk") depends on several factors, the most common being the degree of disease at the time of first treatment.
"neoadjuvant setting" refers to the clinical setting in which the method is performed prior to primary/definitive therapy.
As used herein, "delaying" the progression of cancer means delaying, impeding, slowing, delaying, stabilizing and/or delaying the progression of the disease. Such delays may have varying lengths of time depending on the medical history and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may actually encompass prevention, as the individual does not develop the disease. A method of "delaying" the progression of cancer is a method of reducing the probability of disease progression and/or reducing the extent of disease within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects. Cancer progression may be detectable by standard methods including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, coagulation tests, arteriography, biopsy, urine cytology, and cystoscopy. Progression may also refer to cancer progression that may not be detectable initially, and includes occurrence, recurrence and onset.
The term "effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a given disorder, condition, or disease, such as to ameliorate, reduce, and/or delay one or more symptoms thereof. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and/or to reduce the rate of tumor growth (such as to inhibit tumor growth) or to prevent or delay other undesirable cell proliferation in the cancer. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay relapse. In some embodiments, an effective amount is an amount sufficient to reduce the rate of relapse in an individual. An effective amount may be administered in one or more administrations. The effective amount of the drug or composition may: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibit, retard, slow down, and preferably stop cancer cell infiltration to some extent into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the occurrence and/or recurrence of tumors; (vii) (viii) reducing the rate of recurrence of the tumor, and/or (viii) alleviating to some extent one or more symptoms associated with the cancer.
As understood in the art, an "effective amount" may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired therapeutic endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and if a desired or beneficial result is possible or achieved in combination with one or more other agents, administration of the therapeutic agent (e.g., an antibody-drug conjugate) in an effective amount may be considered. The components of the combination therapies of the invention (e.g., the first and second therapies) can be administered sequentially, simultaneously, or concurrently using the same or different routes of administration for each component. Thus, an effective amount of a combination therapy includes an amount of the first therapy and an amount of the second therapy that, when administered sequentially, simultaneously, or concurrently, produces the desired result.
An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets (such as cats, dogs, horses), primates, mice, and rats.
The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.
As used herein, "full length antibody" refers to a molecule comprising the native biological form of an antibody, including the variable and constant regions. For example, in most mammals (including humans and mice), full length antibodies of the IgG isotype are tetrameric and consist of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2, and CH 3. In some mammals, for example in camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable domain attached to an Fc region.
As used herein, "Fc region" refers to a polypeptide that comprises the constant region of an antibody heavy chain and excludes the first constant region immunoglobulin domain. For IgG, the Fc region may comprise the immunoglobulin domains CH2 and CH3 and the hinge between CH1 and CH 2.
As used herein, the term "specific recognition" or "specific binding" refers to a measurable and reproducible interaction, such as attraction or binding between a target and an antibody (or molecule or moiety), which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically or preferentially binds an epitope is one that binds to the target in a specific mannerOther epitopes or non-target epitopes bind antibodies that bind to the epitope with greater affinity, avidity, more readily and/or for longer durations. It is also understood that, for example, an antibody (or portion or epitope) that specifically or preferentially binds a first target may or may not specifically or preferentially bind a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. An antibody that specifically binds a target can have at least about 10 3 M -1 Or 10 4 M -1 Sometimes about 10 5 M -1 Or 10 6 M -1 About 10 in other cases 6 M -1 Or 10 7 M -1 About 10 8 M -1 To 10 9 M -1 Or about 10 10 M -1 To 10 11 M -1 Or higher association constants. Various immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to select monoclonal antibodies specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity, see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.
The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin (variable domains) that contain an antigen binding site. The constant domains comprise the CH1, CH2, and CH3 domains of the heavy chain (collectively referred to as CH) and the CHL (or CL) domain of the light chain.
The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site.
The term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed among the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs, also called CDRs) which are both in the light and heavy chain variable domains. The most highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, predominantly in a β -sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the β -sheet structure. The HVRs in each chain are closely linked together by the FR region and contribute to the formation of the antigen binding site of the antibody with HVRs from the other chain (see Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.
The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinctly different classes, termed kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.
The term IgG "isotype" or "subclass" as used herein means any immunoglobulin subclass defined by the chemical and antigenic characteristics of its constant regions. Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, γ, ɛ, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al Cellular and mol. The antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
As used herein, the term "CDR" or "complementarity determining region" is intended to mean a non-contiguous antigen combining site found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described in Kabat et Al, J.biol. chem. 252:6609-6616 (1977), Kabat et Al, U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991), Chothia et Al, J.mol. biol. 196:901-917 (1987), Al-Lazikani B. et Al,J. Mol. Biol.273: 927-,Mol. Immunol.,45: 3832-,Dev. Comp. Immunol.27: 55-77 (2003), and Honegger and Pluckthun,J. Mol. Biol.309:657-670 (2001), wherein the definition includes an overlap or subset of amino acid residues when compared against each other. Notwithstanding, it is intended that any definition applied to refer to a CDR or variant thereof of an antibody or grafted antibody is within the scope of the term as defined and used herein. Amino acid residues encompassing the CDRs as defined by each of the references cited above are set forth in table a below for comparison. CDR prediction algorithms and interfaces are known in the art and include, for example, Abhinandan and Martin,Mol. Immunol.,45: 3832-3839 (2008), Ehrenmann F. et al,Nucleic Acids Res.38D 301-D307 (2010), and Adolf-Bryfogle J, et al,Nucleic Acids Res.43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated by reference herein in their entirety for use in the present invention and for possible inclusion in one or more claims herein.
Table a: CDR definition
Kabat 1 Chothia 2 MacCallum 3 IMGT 4 AHo 5
V H CDR1 31-35 26-32 30-35 27-38 25-40
V H CDR2 50-65 53-55 47-58 56-65 58-77
V H CDR3 95-102 96-101 93-101 105-117 109-137
V L CDR1 24-34 26-32 30-36 27-38 25-40
V L CDR2 50-56 50-52 46-55 56-65 58-77
V L CDR3 89-97 91-96 89-96 105-117 109-137
1 Residue numbering follows the Kabat et al, supra nomenclature
2 Residue numbering follows the nomenclature of Chothia et al, supra
3 Residue numbering follows the nomenclature of MacCallum et al, supra
4 Residue numbering follows the nomenclature of Lefranc et al, supra
5 Residue numbering follows the nomenclature of Honegger and Pluckthun, supra.
"percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences, taking into account any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity may be performed in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For purposes herein, however, the sequence comparison computer program MUSCLE (Edgar, r.c.,Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics5(1) 113, 2004) to generate amino acid sequence identity% values.
The "CH 1 domain" of the human IgG Fc region (also referred to as "C1" of the "H1" domain) typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).
A "hinge region" is generally defined as extending from Glu216 of human IgG1 to Pro230 (Burton,Molec. Immunol.22:161-206 (1985)). The hinge region of other IgG isotypes can be aligned to the IgG1 sequence by placing the first and last cysteine residues that form the S-S bond between heavy chains at the same position.
The "CH 2 domain" of the human IgG Fc region (also referred to as "C2" of the "H2" domain) typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. In contrast, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native IgG molecule. It has been speculated that carbohydrates may provide an alternative to domain-domain pairings and help stabilize the CH2 domain. The number of the Burton is equal to the number of the Burton,Molec Immunol. 22:161-206 (1985)。
the "CH 3 domain" (also referred to as the "C2" or "H3" domain) comprises an extension of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to the C-terminal of the antibody sequence (typically at amino acid residue 446 or 447 of an IgG)).
By "amino acid modification" herein is meant amino acid substitutions, insertions and/or deletions in the polypeptide sequence. By "amino acid substitution" or "substitution" herein is meant the replacement of an amino acid at a given position in a protein sequence with another amino acid. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide (typically a native or "parent" polypeptide). The polypeptide variants may have one or more amino acid substitutions, deletions and/or insertions at certain positions within the native amino acid sequence.
"transglutaminase" is used interchangeably herein with "TGase" and refers to an enzyme capable of performing a transglutaminase reaction. The term "transglutaminase" as used herein refers to a reaction wherein gamma-glutaminyl groups from acceptor glutamine residues of proteins/peptides are transferred to an amine group, such as the epsilon-amino group of a primary amine or lysine.
The term "acceptor glutamine residue", when referring to an amino acid residue of a polypeptide or protein, refers to a glutamine residue that is recognized by TGase under suitable conditions and can be cross-linked to a conjugate moiety comprising a donor amine group by TGase via a reaction between glutamine and the donor amine group (such as lysine or a structurally related primary amine such as an amino pentyl group).
As used herein, "endogenous acceptor glutamine residues on an antibody" refers to acceptor glutamine residues in the Fc region of a naturally occurring antibody. In some embodiments, the endogenous acceptor glutamine residue is Q295 by EU numbering and is flanked by an N-glycosylation site at position Asn 297.
It is to be understood that the aspects and embodiments of the invention described herein include "consisting of and" consisting essentially of aspects and embodiments.
Reference herein to a "value or parameter" about "includes (and describes) variations that are directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X". The term "about X-Y" as used herein has the same meaning as "about X to about Y".
As used herein, reference to "not" a value or parameter generally means and describes "in addition to a value or parameter.
As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
Methods of treatment
The present application provides methods of treating cancer, such as HER 2-positive cancer, using antibody-drug conjugates (ADCs) comprising an anti-HER 2 antibody conjugated via an endogenous receptor glutamine residue in the Fc region of the anti-HER 2 antibody to a conjugate moiety comprising a toxin. Any of the ADCs described in section III "antibody-drug conjugates (ADCs)" may be used in the methods described herein.
In some embodiments, there is provided a method of treating HER 2-positive (e.g., HER 2+ or HER 23 +) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, and wherein the conjugate moiety is conjugated to the receptor glutamine residue. In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at the +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of the heavy chain of the anti-HER 2 antibody, wherein the numbering is according to EU numbering. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, provided is a method of treating HER 2-positive (e.g., HER 2+ or HER 23 +) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising endogenous receptor glutamine residues, wherein the conjugate moiety comprises monomethyl auristatin e (mmae), and wherein the anti-HER 2 antibody is conjugated to the conjugate moiety via the receptor glutamine residues. In some embodiments, the conjugate moiety has the chemical structure of formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, provided are methods of treating HER 2-positive (e.g., HER 2+ or HER 23 +) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: (a) a VH comprising: HC-CDRL comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID No. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID No. 5 and LC-CDR3 comprising the amino acid sequence of SEQ ID No. 6, wherein said anti-HER 2 antibody comprises a glycosylated (e.g. N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein said conjugate moiety has the chemical structure of formula (III), wherein N is an integer between 1 and 12, and wherein said conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO:7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, provided are methods of treating HER 2-positive (e.g., HER 2+ or HER 23 +) cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10, wherein the conjugate moiety has the chemical structure of formula (I), and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via an acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the Fc region of the anti-HER 2 antibody is N-glycosylated at position 297, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the anti-HER 2 antibody is DP 001. In some embodiments, the anti-HER 2 antibody is trastuzumab. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, there is provided a method of treating a HER 2-positive cancer that is resistant or refractory to a second HER2-targeting agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, and wherein the conjugate moiety is conjugated to the receptor glutamine residue. In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at the +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of the heavy chain of the anti-HER 2 antibody, wherein the numbering is according to EU numbering. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the second HER2 targeting agent is trastuzumab, enrituril trastuzumab, pertuzumab, or lapatinib. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, there is provided a method of treating a HER 2-positive cancer that is resistant or refractory to a second HER2-targeting agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising endogenous acceptor glutamine residues, wherein the conjugate moiety comprises MMAE, and wherein the anti-HER 2 antibody is conjugated to the conjugate moiety via the acceptor glutamine residues. In some embodiments, the conjugate moiety has the chemical structure of formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the second HER2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary tract, or lung cancer.
In some embodiments, there is provided a method of treating a HER 2-positive cancer that is resistant or refractory to a second HER2-targeting agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: (a) a VH comprising: HC-CDRL comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID No. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID No. 5 and LC-CDR3 comprising the amino acid sequence of SEQ ID No. 6, wherein said anti-HER 2 antibody comprises a glycosylated (e.g. N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein said conjugate moiety has the chemical structure of formula (III), wherein N is an integer between 1 and 12, and wherein said conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO:7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the second HER2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, there is provided a method of treating a HER 2-positive cancer that is resistant or refractory to a second HER2-targeting agent in an individual, comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10, wherein the conjugate moiety has the chemical structure of formula (I), and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via an acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the Fc region of the anti-HER 2 antibody is N-glycosylated at position 297, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the anti-HER 2 antibody is DP 001. In some embodiments, the anti-HER 2 antibody is trastuzumab. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the second HER2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib. In some embodiments, the HER 2-positive cancer is a solid cancer, such as breast, colorectal, ovarian, gastric, urinary or lung cancer.
In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety is conjugated to the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at the +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295 of the heavy chain of the anti-HER 2 antibody, wherein numbering is according to EU numbering. In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg, such as about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2mg/kg to about 3 mg/kg, such as about 2.0 mg/kg or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once a week. In some embodiments, the cancer is a HER 2-positive cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety comprises MMAE, wherein the anti-HER 2 antibody is conjugated to the conjugate moiety via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg (e.g., no more than about 6 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the conjugate moiety has the chemical structure of formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg, such as about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2mg/kg to about 3 mg/kg, such as about 2.0 mg/kg or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once a week. In some embodiments, the cancer is a HER 2-positive cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: (a) a VH comprising: HC-CDRL comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a VL comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID No. 4, an LC-CDR2 comprising the amino acid sequence of SEQ ID No. 5, and an LC-CDR3 comprising the amino acid sequence of SEQ ID No. 6, wherein the anti-HER 2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety has the chemical structure of formula (III), wherein N is an integer between 1 and 12, wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of NO more than about 8 mg/kg (e.g., NO more than about 6 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the Fc region of the anti-HER 2 antibody is N-glycosylated at position 297, wherein the numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO:7, and (b) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg, such as about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2mg/kg to about 3 mg/kg, such as about 2.0 mg/kg or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once a week. In some embodiments, the cancer is a HER 2-positive cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID No. 9 and a light chain comprising the amino acid sequence of SEQ ID No. 10, wherein the conjugate moiety has the chemical structure of formula (I), wherein the conjugate moiety is conjugated to an anti-HER 2 antibody via an acceptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of NO more than about 8 mg/kg (e.g., NO more than about 6 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the Fc region of the anti-HER 2 antibody is N-glycosylated at position 297, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the anti-HER 2 antibody is DP 001. In some embodiments, the anti-HER 2 antibody is trastuzumab. In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg, such as about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2mg/kg to about 3 mg/kg, such as about 2.0 mg/kg or about 3.0 mg/kg. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered about once every three weeks, about every other week, or about once a week. In some embodiments, the cancer is a HER 2-positive cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein DP303c is administered intravenously about once every three weeks at a dose of no more than about 8 mg/kg. In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg. In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein DP303c is administered intravenously about once every three weeks at a dose of about 1mg/kg to about 2 mg/kg. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of DP303c, wherein DP303c is administered intravenously about once every three weeks at a dose of about 2mg/kg to about 3 mg/kg. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg or about 3.0 mg/kg. In some embodiments, the cancer is a HER 2-positive cancer. In some embodiments, the cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer. In some embodiments, the HER 2-positive cancer is HER 23 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is HER 22 + as determined by an IHC test. In some embodiments, the HER 2-positive cancer is positive as determined by a FISH test. In some embodiments, the subject is non-responsive or not suitable for standard therapy. In some embodiments, the individual has not previously received a second HER2-targeting agent. In some embodiments, the individual has previously received a second HER2-targeting agent. In some embodiments, the cancer is resistant or refractory to a second HER2-targeting agent. In some embodiments, the second HER2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib.
DP303c is an antibody drug conjugate with a monoclonal IgG1 antibody (DP001) targeting HER2 and one cleavable LND1002 (toxin) site-specifically conjugated to glutamine 295 in the constant region of each heavy chain of DP 001. DP303c has a DAR (drug-antibody ratio) of about 1.8 to about 2.2, such as about 1.8, 1.9, 2.0, 2.1 or 2.2.
Cancer treatment can be assessed by, for example, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Methods of determining the efficacy of the therapy may be employed, including measuring the response, for example, by radiographic imaging.
Exemplary routes of administration of the ADC include, but are not limited to, oral, intravenous, intracavity, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or delivered into lymph glands, body spaces, organs, or tissues known to contain cancer cells. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered by infusion. In some embodiments, the ADC is administered by injection.
The dosage regimen of the ADC administered to an individual may vary with the particular ADC composition, method of administration, and the particular type and stage of cancer being treated. In some embodiments, the effective amount of ADC is below a level that induces a toxicological effect (i.e., an effect above a clinically acceptable toxicity level) or at a level that can control or tolerate potential side effects when the composition is administered to an individual. The doses referred to herein are determined relative to the overall molecular weight of the ADC. In some embodiments, the ADC is administered at a dose of no more than about any one of: 12mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg or 0.3 mg/kg. In some embodiments, the dosage of the ADC is within any one of the following ranges, wherein the range has an upper limit of any one of: 12mg/kg, 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg or 0.4 mg/kg, and an independently selected lower limit of any one of: 11 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7.5 mg/kg, 7 mg/kg, 6.5 mg/kg, 6 mg/kg, 5.5 mg/kg, 5 mg/kg, 4.5 mg/kg, 4 mg/kg, 3.5 mg/kg, 3.25 mg/kg, 3 mg/kg, 2.9 mg/kg, 2.8 mg/kg, 2.75 mg/kg, 2.7 mg/kg, 2.6 mg/kg, 2.5 mg/kg, 2.4 mg/kg, 2.3 mg/kg, 2.25 mg/kg, 2.2 mg/kg, 2.1 mg/kg, 2mg/kg, 1.9 mg/kg, 1.8 mg/kg, 1.75 mg/kg, 1.7 mg/kg, 1.6 mg/kg, 1.5 mg/kg, 1.4 mg/kg, 1.3 mg/kg, 1.25 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 1mg/kg, 0.8 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg or 0.3 mg/kg and wherein the lower limit is less than the upper limit. In some embodiments, the ADC is administered at a dose of any one of: about 0.3 mg/kg to about 12mg/kg, about 0.6 mg/kg to about 8 mg/kg, about 1mg/kg to about 8 mg/kg, about 3 mg/kg to about 8 mg/kg, about 0.6 mg/kg to about 6 mg/kg, about 1mg/kg to about 2mg/kg, about 1mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 2.0 mg/kg, about 2.0 mg/kg to about 2.5 mg/kg, about 1mg/kg to about 3 mg/kg, about 2mg/kg to about 3 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 2mg/kg to about 2.5 mg/kg, about 1.5 mg/kg to about 2.5 mg/kg, From about 0.5 mg/kg to about 3.0 mg/kg, from about 2mg/kg to about 4 mg/kg, from about 4 mg/kg to about 8 mg/kg, from about 8 mg/kg to about 12mg/kg, from about 0.5 mg/kg to about 5 mg/kg, or from about 1mg/kg to about 5 mg/kg. The dosage as described herein may refer to a suitable dosage for cynomolgus monkeys, a human equivalent thereof, or an equivalent dosage for a particular species of subject. In some embodiments, for a cynomolgus monkey or a human subject, the ADC is administered at a dose equivalent to about 0.3 mg/kg to about 8 mg/kg (such as, e.g., about 0.3 mg/kg to about 6 mg/kg, about 0.6 mg/kg to about 4.5 mg/kg, about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, for a cynomolgus monkey or a human subject, the ADC is administered at a dose equivalent to no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg, or about 2 mg/kg; or from about 1mg/kg to about 2mg/kg, or from about 2mg/kg to about 3 mg/kg).
In some embodiments, the ADC is administered at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3, 0.6, 1, 2, 3, 4.5, 6, or 8 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg, such as about 1.0 mg/kg or about 2.0 mg/kg. In some embodiments, the ADC is administered at a dose of about 2mg/kg to about 3 mg/kg, such as about 2.0 mg/kg or about 3.0 mg/kg.
An effective amount of ADC may be administered in a single dose or multiple doses. For methods that include administering the ADC in multiple doses, exemplary dosing frequencies include, but are not limited to, once per week without interruption, once per week for two of three weeks, once per week for three of four weeks, once per three weeks, once per two weeks, once per month, once per six months, once per year, and the like. In some embodiments, the ADC is administered about once per week, once every 2 weeks, or once every 3 weeks. In some embodiments, the interval between each administration is less than any one of about 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week. In some embodiments, the interval between each administration is greater than any one of about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, the dosing schedule is not interrupted.
In some embodiments, the MSFP is administered at a low frequency, e.g., no more than any one of weekly, every other week, every three weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every year, or less. In some embodiments, the ADC is administered in a single dose. In some embodiments, the ADC is administered about once every three weeks.
In some embodiments, the ADC is administered at a dose of no more than about 8 mg/kg, such as no more than any of 6 mg/kg, 4.5 mg/kg, 3 mg/kg, 2mg/kg, or 1mg/kg, once a week, once every other week, or once every three weeks. In some embodiments, the ADC is administered weekly, every other week, or every three weeks at a dose of any one of about 0.3 mg/kg to about 8 mg/kg, such as about 0.3, 0.6, 1, 2, 3, 4.5, 6, or 8 mg/kg. In some embodiments, the ADC is administered at a dose of about 1mg/kg to about 2mg/kg once a week, once every other week, or once every three weeks. In some embodiments, the ADC is administered weekly, every other week, or every three weeks at a dose of about 2mg/kg to about 3 mg/kg. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg once a week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg once a week, once every other week, or once every three weeks. In some embodiments, the ADC is administered at a dose of about 3.0 mg/kg once a week, once every other week, or once every three weeks.
The administration of the ADC may be extended for an extended period of time, such as from about one week to about one month, from about one month to about one year, from about one year to about several years. In some embodiments, the MSFP is administered over a period of at least about any one of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or longer.
The methods described herein are useful for treating various cancers, such as solid cancers. The methods are applicable to all stages of cancer, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. In an adjuvant setting or neoadjuvant setting, the methods described herein may be used as a first therapy, a second therapy, a third therapy, or a combination therapy with other types of cancer therapies known in the art (such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, and the like). In some embodiments, the cancer is resistant or refractory to a previous therapy. In some embodiments, the subject has relapsed from a previous therapy. In some embodiments, the individual has recurrent cancer. In some embodiments, the method is performed prior to an initial/definitive therapy. In some embodiments, the method is for treating an individual who has been previously treated. In some embodiments, the method is for treating an individual who has not been previously treated. In some embodiments, the method is used as a first line therapy. In some embodiments, the method is used as a second line therapy.
In some embodiments, the methods are suitable for treating cancers that overexpress HER2 on the surface of cancer cells, such as HER 2-positive solid cancers. In some embodiments, the solid cancer is HER 22 + as determined by an IHC test. In some embodiments, the solid cancer is HER 23 + as determined by an IHC test. In some embodiments, the solid cancer is e.g., tongHER2 positive as determined by FISH testing. In some embodiments, the cancer cells in the individual express at least about more than 2,5, 10, 20, 50, 100, 200, 500, 1000 or more times any of HER2 as compared to normal cells. In some embodiments, the cancer cells in the individual have no more than about 250,000, such as no more than about 200,000; 100,000; 75,000; 50,000; 25,000; 10,000; 7,500; or 5,000 relative HER2 density as determined using quantitative HER2 receptor densitometry, such as Quantum 647 mesf (bang laboratories). In some embodiments, the cancer cells in the individual have a HER2 receptor density comparable to or greater than JIMT-1 cells. HER2 density has been described for various cancer cell lines, see, e.g., LI. JY et al, "A biocompatible HER2-targeting antagonist-drug conjugate indexes growth in primary modules recovery to or inorganic for HER2-targeted therapy"Cancer Cell29(1) — 117-. In some embodiments, the HER 2-positive solid cancer is selected from breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
In some embodiments, there is provided a method of treating breast cancer (e.g., HER 2-positive breast cancer) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the breast cancer is early breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, remitted breast cancer, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is in a neoadjuvant setting. In some embodiments, the breast cancer is in advanced stages. In some embodiments, the breast cancer is HER 2-positive breast cancer. In some embodiments, the breast cancer is HER 22 + as determined by an IHC test. In some embodiments, the breast cancer is HER 23 + as determined by an IHC test. In some embodiments, the breast cancer is HER2 positive as determined by FISH testing. In some embodiments, the breast cancer has metastasized to the liver, lung, adrenal glands, lymph nodes, and/or peritoneum.
In some embodiments, there is provided a method of treating ovarian cancer (e.g., HER 2-positive ovarian cancer) in an individual comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the ovarian cancer is an ovarian epithelial cancer. In some embodiments, the ovarian cancer is stage I (e.g., IA, IB, or IC), stage II (e.g., IIA, IIB, or IIC), stage III (e.g., IIIA, HIB, or HIC), or stage IV. In some embodiments, the ovarian cancer is HER 2-positive ovarian cancer. In some embodiments, the ovarian cancer is HER 22 + as determined by an IHC test. In some embodiments, the ovarian cancer is HER 23 + as determined by an IHC test. In some embodiments, the ovarian cancer is HER2 positive as determined by the FISH test.
In some embodiments, there is provided a method of treating colorectal cancer (e.g., HER 2-positive colorectal cancer) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the colorectal cancer is a sigmoid colon cancer. In some embodiments, the colorectal cancer is stage I, II (e.g., IIA, IIB, or IIC), III (e.g., IIIA, IIIB, or IIIC), or IV (e.g., IVA, IVB, or IVC). In some embodiments, the ovarian cancer is HER 2-positive ovarian cancer. In some embodiments, the colorectal cancer is HER 22 + as determined by an IHC test. In some embodiments, the colorectal cancer is HER 23 + as determined by an IHC test. In some embodiments, the colorectal cancer is HER2 positive as determined by a FISH test.
In some embodiments, there is provided a method of treating gastric cancer (e.g., HER 2-positive gastric cancer) in an individual comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the gastric cancer is adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), or carcinoid tumor. In some embodiments, the gastric cancer is stage 0 (carcinoma in situ), stage I, stage II, stage III, or stage IV. In some embodiments, the gastric cancer is HER 2-positive gastric cancer. In some embodiments, the gastric cancer is HER 22 + as determined by IHC testing. In some embodiments, the gastric cancer is HER 23 + as determined by IHC testing. In some embodiments, the gastric cancer is HER2 positive as determined by FISH testing.
In some embodiments, there is provided a method of treating urinary tract cancer (e.g., HER 2-positive urinary tract cancer) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the urethral carcinoma is a squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma. In some embodiments, the urethral cancer is a distal urethral cancer or a proximal urethral cancer. In some embodiments, the urethral cancer is HER 2-positive urethral cancer. In some embodiments, the urethral cancer is HER 22 + as determined by the IHC test. In some embodiments, the urethral carcinoma is HER 23 + as determined by an IHC test. In some embodiments, the urinary tract cancer is HER2 positive as determined by the FISH test.
In some embodiments, there is provided a method of treating lung cancer (e.g., HER 2-positive lung cancer) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large cell carcinoma, adenocarcinoma, neuroendocrine lung tumor, and squamous cell carcinoma. In some embodiments, the lung cancer is Small Cell Lung Cancer (SCLC). In some embodiments, the lung cancer is HER 2-positive lung cancer. In some embodiments, the lung cancer is HER 22 + as determined by an IHC test. In some embodiments, the lung cancer is HER 23 + as determined by an IHC test. In some embodiments, the lung cancer is HER2 positive as determined by FISH testing.
The methods described herein may be used in various aspects of cancer treatment. In some embodiments, there is provided a method of inhibiting cell proliferation (such as tumor growth) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited.
In some embodiments, there is provided a method of inhibiting tumor metastasis in an individual comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis is inhibited.
In some embodiments, there is provided a method of reducing (such as eradicating) pre-existing tumor metastasis (such as metastasis to a lymph node) in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or from about 1mg/kg to about 2mg/kg, or from about 2mg/kg to about 3 mg/kg). In some embodiments, at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) of the metastasis is reduced.
In some embodiments, there is provided a method of reducing the incidence or burden of pre-existing tumor metastasis (such as metastasis to lymph nodes) in a subject, comprising administering to the subject an effective amount of any of the ADCs described herein, wherein the ADC is administered to the subject at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg).
In some embodiments, there is provided a method of reducing tumor size in an individual comprising administering to the individual an effective amount of any of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg).
In some embodiments, there is provided a method of increasing the time to progression of a cancer disease in an individual, comprising administering to the individual an effective amount of any of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the method extends the time to disease progression by at least any one of 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36 or more weeks.
In some embodiments, there is provided a method of prolonging the survival of an individual having cancer, comprising administering to the individual an effective amount of any one of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg). In some embodiments, the method extends the survival of the individual by at least any one of 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 18, or 24 months.
In some embodiments, there is provided a method of alleviating one or more symptoms in an individual having cancer, comprising administering to the individual an effective amount of any of the ADCs described herein, wherein the ADC is administered to the individual at a dose of no more than about 8 mg/kg (such as no more than about 6 mg/kg, about 4.5 mg/kg, about 3 mg/kg or about 2 mg/kg; or about 1mg/kg to about 2mg/kg, or about 2mg/kg to about 3 mg/kg).
The invention also provides a composition of any one of the ADCs described herein for use in the methods described in this section and the use of the ADC in the manufacture of a medicament for the treatment of cancer.
Antibody-drug conjugates (ADC)
The present application also provides antibody-drug conjugates (ADCs) useful in the therapeutic methods described herein. The ADC may comprise any of the anti-HER 2 antibodies described herein conjugated to any of the conjugate moieties described herein via an endogenous receptor glutamine residue in the Fc region of the anti-HER 2 antibody.
In some embodiments, an ADC is provided comprising an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugate moiety is conjugated to the acceptor glutamine residue. In some embodiments, the Fc region is N-glycosylated. In some embodiments, the acceptor glutamine residue is flanked by N-glycosylation sites at the +2 position relative to the glutamine residue.
In some embodiments, an ADC is provided comprising a full length anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via the receptor glutamine residue at position 295 of the heavy chain of the anti-HER 2 antibody, wherein numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody comprises an N-glycosylated Fc region. In some embodiments, the conjugate moiety is conjugated to the anti-HER 2 antibody via an acceptor glutamine residue at position 295 of a heavy chain of the anti-HER 2 antibody, and wherein N-glycosylation is at position 297 of the heavy chain, wherein numbering is according to EU numbering.
In some embodiments, an ADC is provided comprising an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety comprises at least one (such as 1, 2, or more) MMAE, wherein the anti-HER 2 antibody is conjugated to the conjugate moiety via the acceptor glutamine residue. In some embodiments, the conjugate moiety has the chemical structure of formula (II), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (III), wherein n is an integer between 1 and 12. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the average molar ratio between the conjugating moiety and the anti-HER 2 antibody in the composition is about 1:1 to about 2: 1. In some embodiments, the molar ratio of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of ADCs in the composition between the conjugate moiety and the anti-HER 2 antibody is about 2: 1.
In some embodiments, there is provided an ADC comprising an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: (a) a VH comprising: HC-CDRL comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID No. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID No. 5 and LC-CDR3 comprising the amino acid sequence of SEQ ID No. 6, wherein said anti-HER 2 antibody comprises a glycosylated (e.g. N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein said conjugate moiety has the chemical structure of formula (III), wherein N is an integer between 1 and 12, and wherein said conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the average molar ratio between the conjugating moiety and the anti-HER 2 antibody in the composition is about 1:1 to about 2: 1. In some embodiments, the molar ratio of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of ADCs in the composition between the conjugate moiety and the anti-HER 2 antibody is about 2: 1.
In some embodiments, there is provided an ADC comprising an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: (a) VH comprising the amino acid sequence of SEQ ID NO 7; and (b) a VL comprising the amino acid sequence of SEQ ID NO:8, wherein the anti-HER 2 antibody comprises a glycosylated (e.g., N-glycosylated) Fc region comprising an endogenous acceptor glutamine residue, wherein the conjugate moiety has the chemical structure of formula (III) wherein N is an integer between 1 and 12, and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the anti-HER 2 antibody is N-glycosylated at position 297 of the Fc region, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the average molar ratio between the conjugation moiety and the anti-HER 2 antibody in the composition is about 1:1 to about 2: 1. In some embodiments, the molar ratio of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of ADCs in the composition between the conjugate moiety and the anti-HER 2 antibody is about 2: 1.
In some embodiments, there is provided an ADC comprising an anti-HER 2 antibody and a conjugate moiety, wherein the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID No. 9 and a light chain comprising the amino acid sequence of SEQ ID No. 10, wherein the conjugate moiety has the chemical structure of formula (III), wherein n is an integer between 1 and 12, and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via an acceptor glutamine residue. In some embodiments, the acceptor glutamine residue is at position 295, wherein numbering is according to EU numbering. In some embodiments, the Fc region of the anti-HER 2 antibody is N-glycosylated at position 297, wherein the numbering is according to EU numbering. In some embodiments, the conjugate moiety has the chemical structure of formula (I). In some embodiments, the conjugate moiety is LND 1002. In some embodiments, the anti-HER 2 antibody is DP 001. In some embodiments, the anti-HER 2 antibody is trastuzumab. In some embodiments, the ADC is DP303 c. In some embodiments, the average molar ratio between the conjugating moiety and the anti-HER 2 antibody in the composition is about 1:1 to about 2: 1. In some embodiments, the molar ratio of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of ADCs in the composition between the conjugate moiety and the anti-HER 2 antibody is about 2: 1.
In some embodiments, the anti-HER 2 antibody is a full length antibody. In some embodiments, the anti-HER 2 antibody is an antibody fragment comprising an Fc region. In some embodiments, the Fc region comprises part or all of the hinge region. In some embodiments, the anti-HER 2 antibody comprises an Fc region of a naturally occurring immunoglobulin. In some embodiments, the anti-HER 2 antibody comprises an IgG1, IgG2, IgG3, IgG4 subtype or an Fc region from IgA, IgE, IgD, or IgM. In some embodiments, the Fc region is from a human IgG and is from the amino acid residue at position Glu216 or Ala231 to its carboxy terminus according to the EU numbering system.
In some embodiments, the Fc region in the anti-HER 2 antibody is N-glycosylated. For example, in some embodiments, the polysaccharide chain attached at an N-glycosylation site is at least about any one of 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units.
In some embodiments, provided are compositions comprising any of the ADCs described herein, wherein at least some (but not necessarily all) of the anti-HER 2 antibodies in the composition are glycosylated (e.g., N-glycosylated) in the Fc region. For example, in some embodiments, compositions are provided comprising an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody conjugated to a conjugate moiety via an endogenous acceptor glutamine residue on the anti-HER 2 antibody, wherein at least some (e.g., at least any one of about 50%, 60%, 70%, 80%, 90%, or 95%) of the antibody-drug conjugate in the composition is glycosylated (e.g., N-glycosylated) in the Fc region.
In some embodiments, the N-glycosylation site flanks the acceptor glutamine residue to which the conjugate moiety is conjugated. In some embodiments, the N-glycosylation site and the acceptor glutamine residue are separated by 5 or lessAnd (c) amino acid residues. In some embodiments, the N-glycosylation site and the acceptor glutamine are separated by 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the N-glycosylation site and the acceptor glutamine are adjacent to each other. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at the +2 position relative to the glutamine residue. In some embodiments, the acceptor glutamine residue is flanked at position +1, +2, +3, +4, or +5 relative to the glutamine residue by an N-glycosylation site. In some embodiments, the acceptor glutamine residue is flanked by an N-glycosylation site at position-1, -2, -3, -4, or-5 relative to the glutamine residue. In some embodiments, the N-glycosylated Fc region comprises SEQ ID NO 11 (KPREEQX) 1 NSTX 2 R, wherein X 1 Is Y or F and X 2 Is Y or F), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 6 of SEQ ID NO:11, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 11. In some embodiments, the N-glycosylated Fc region comprises the amino acid sequence of SEQ ID NO:12 (KPREEQYNSTYR), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 6 of SEQ ID NO:12, and wherein the N-glycosylation is at position 8 of SEQ ID NO: 2.
In some embodiments, the anti-HER 2 antibody comprises an Fc region of human IgG 1. In some embodiments, the anti-HER 2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO:13 (the CH2 sequence of human IgG1), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 65 of SEQ ID NO:13, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 13.
In some embodiments, the anti-HER 2 antibody comprises an Fc region of human IgG 2. In some embodiments, the anti-HER 2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO:14 (the CH2 sequence of human IgG2), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 64 of SEQ ID NO:14, and wherein the N-glycosylation is at position 66 of SEQ ID NO: 14.
In some embodiments, the anti-HER 2 antibody comprises an Fc region of human IgG 3. In some embodiments, the anti-HER 2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO:15 (the CH2 sequence of human IgG3), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 65 of SEQ ID NO:15, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 15.
In some embodiments, the anti-HER 2 antibody comprises an Fc region of human IgG 4. In some embodiments, the anti-HER 2 antibody comprises an N-glycosylated Fc region comprising the amino acid sequence of SEQ ID NO:16 (the CH2 sequence of human IgG4), and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via an acceptor glutamine residue at position 65 of SEQ ID NO:16, and wherein the N-glycosylation is at position 67 of SEQ ID NO: 16.
SEQ ID NO 13 (human IgG1)
Figure 417DEST_PATH_IMAGE003
SEQ ID NO 14 (human IgG2)
Figure 130047DEST_PATH_IMAGE004
SEQ ID NO 15 (human IgG3)
Figure 33500DEST_PATH_IMAGE005
16 (human IgG4)
Figure 387121DEST_PATH_IMAGE006
The ADCs described herein have an anti-HER 2 antibody component conjugated to a conjugate moiety in a specific and stoichiometrically controlled manner (i.e., at the acceptor glutamine residue of the Fc region flanked by N-glycosylation sites). In some embodiments, the molar ratio of the conjugate moiety to the anti-HER 2 antibody is about 1: 1. In some embodiments, the molar ratio of the conjugate moiety to the anti-HER 2 antibody is about 2: 1. In some embodiments, the molar ratio of conjugate moieties of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of the ADCs in the composition to anti-HER 2 antibody is about 1: 1. In some embodiments, the molar ratio of conjugate moieties of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) of the ADCs to anti-HER 2 antibody in the composition is about 2: 1. In some embodiments, the molar ratio of conjugate moieties of the ADC of at least about 80% (such as at least any of about 85%, 90%, 95%, or more) to anti-HER 2 antibody in the composition is about 1:1 to about 2: 1.
In some embodiments, the ADC is present in an individual (e.g., a mammal) at about 50% or more after at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more after in vivo administration. In some embodiments, the ADC is present in an individual (e.g., a mammal) at about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more after at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, or more after in vivo administration. In some embodiments, the subject has an exposure of free toxin (e.g., MMAE) that is no more than about any of 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, or less of the ADC exposure after in vivo administration of the ADC.
anti-HER 2 antibody
The ADCs described herein comprise an anti-HER 2 antibody. In some embodiments, the anti-HER 2 antibody specifically binds to human HER2 expressed on the cell surface of a human cell (e.g., a human cancer cell). In some embodiments, the anti-HER 2 antibody specifically binds to HER2 expressed on the cell surface of a human cancer cell (e.g., a breast cancer cell, an ovarian cancer cell, a gastric cancer cell, a urinary tract cancer cell, or a lung cancer cell).
In some embodiments, the anti-HER 2 antibody is trastuzumab. In some embodiments, the anti-HER 2 antibody is not trastuzumab. In some embodiments, the anti-HER 2 antibody specifically binds to the same epitope in HER2 as trastuzumab. In some embodiments, the anti-HER 2 antibody comprises the same sequences as trastuzumab, e.g., heavy chain CDRs, light chain CDRs, heavy chain variable regions, light chain variable regions, heavy chain and/or light chain sequences. In some embodiments, the anti-HER 2 antibody is a biological analog of trastuzumab. In some embodiments, the anti-HER 2 antibody is DP 001.
In some embodiments, the anti-HER 2 antibody comprises a heavy chain variable region (VH) comprising: heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID No. 1, or a variant thereof comprising up to about 5 (such as any of about 1, 2, 3,4, or 5) amino acid substitutions, HC-CDR2 comprising the amino acid sequence of SEQ ID No. 2, or a variant thereof comprising up to about 5 (such as any of about 1, 2, 3,4, or 5) amino acid substitutions, and HC-CDR3 comprising the amino acid sequence of SEQ ID No. 3, or a variant thereof comprising up to about 5 (such as any of about 1, 2, 3,4, or 5) amino acid substitutions. In some embodiments, the anti-HER 2 antibody comprises a light chain variable region (VL) comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID No. 4, or a variant thereof comprising up to about 5 (such as any of about 1, 2, 3,4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ ID No. 5, or a variant thereof comprising up to about 3 (such as any of about 1, 2, or 3) amino acid substitutions, and an LC-CDR3 comprising the amino acid sequence of SEQ ID No. 6, or a variant thereof comprising up to about 5 (such as any of about 1, 2, 3,4, or 5) amino acid substitutions.
In some embodiments, the anti-HER 2 antibody comprises: a VH comprising: HC-CDRL comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2 and HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and a VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO. 5 and LC-CDR3 comprising the amino acid sequence of SEQ ID NO. 6.
In some embodiments, the anti-HER 2 antibody comprises: a) a VH comprising the amino acid sequence of SEQ ID NO 1, the amino acid sequence of SEQ ID NO 2 and the amino acid sequence of SEQ ID NO 3; and ii) a VL comprising the amino acid sequence of SEQ ID NO. 4, the amino acid sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.
In some embodiments, the anti-HER 2 antibody comprises: a) VH comprising one, two or three CDRs of SEQ ID NO 7, and/or b) V L Comprising one, two or three CDRs of SEQ ID NO 8. In some embodiments, the anti-HER 2 antibody comprises: a) VH comprising HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain variable region of SEQ ID NO. 7, and/or b) VL comprising LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain variable region of SEQ ID NO. 8.
In some embodiments, the anti-HER 2 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 7 or a variant thereof having at least about 80% (including, e.g., at least any of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 7. In some embodiments, the anti-HER 2 antibody comprises a VL comprising the amino acid sequence of SEQ ID No. 8 or a variant thereof having at least about 80% (including, e.g., at least any of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 8.
In some embodiments, the anti-HER 2 antibody comprises: a) VH comprising the amino acid sequence of SEQ ID NO 7; and b) a VL comprising the amino acid sequence of SEQ ID NO 8.
In some embodiments, the anti-HER 2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 9 or a variant thereof having at least about 80% (including, e.g., at least any one of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 9. In some embodiments, the anti-HER 2 antibody comprises a lambda light chain constant region. In some embodiments, the anti-HER 2 antibody comprises a kappa light chain constant region. In some embodiments, the anti-HER 2 antibody comprises a light chain comprising the amino acid sequence of SEQ ID No. 10 or a variant thereof having at least about 80% (including, e.g., at least any of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID No. 10.
In some embodiments, the anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10.
Exemplary anti-HER 2 antibody sequences are shown in table B below. Exemplary CDR sequences were predicted using the IgBLAST algorithm. See, e.g., Ye J. et al, Nucleic Acids Research, 41: W34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for predicting CDR positions in antibody heavy and light chain variable regions, and antibody agents comprising CDRs from the antibodies described herein but based on prediction algorithms other than IgBLAST are within the scope of the invention. Exemplary antibody heavy and light chain variable region sequences according to the International IMMUNOGENETICS INFORMATION System: (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) ® IMGT) is delimited. See, e.g., Lefranc, m. -p. et al, Nucleic Acids res, 43: D413-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. One skilled in the art will recognize that antibody agents comprising VH or VL sequences from the antibodies described herein, but based on algorithms other than IMGT, are within the scope of the invention.
Table b. anti-HER 2 antibody sequences
SEQ ID NO HC-CDR SEQ ID NO LC-CDR
1 GFNIKDTYIH 4 RASQDVNTAVA
2 RIYPTNGYTRYADSVKG 5 SASFLYS
3 WGGDGFYAMDY 6 QQHYTTPPT
SEQ ID NO:7 (VH)
Figure 388575DEST_PATH_IMAGE007
SEQ ID NO:8 (VL)
Figure 180951DEST_PATH_IMAGE008
SEQ ID NO 9 (heavy chain; glycosylation site is bold and underlined)
Figure 318671DEST_PATH_IMAGE009
SEQ ID NO 10 (light chain)
Figure 905510DEST_PATH_IMAGE010
In some embodiments, the composition is prepared byThe anti-HER 2 antibody is DP 001. DP001 is an anti-HER 2 monoclonal antibody having affinity with trastuzumab (HERCEPTIN) ® ) The same amino acid sequence. Specifically, it contains 1328 amino acids with two Heavy Chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO:9) and two Light Chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10). DP001 is a heterotetramer of two HC subclasses of IgG1 and two LC subclasses of κ subclass linked by 16 disulfide bonds (12 intra-and 4 inter-chain). A schematic structure of DP001 is depicted in fig. 1.
The anti-HER 2 antibodies described herein encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab ', F (ab') 2 Fv, Fc, etc.), chimeric antibodies, humanized antibodies, human antibodies (e.g., fully human antibodies), single chain (ScFv), bispecific antibodies, multispecific antibodies, mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity. The antibody may be murine, rat, camelid, human or any other source (including humanized antibodies). Antibodies used in the present disclosure also include single domain antibodies that are either the variable domain of an antibody heavy chain or the variable domain of an antibody light chain. The results of Holt et al,Trends Biotechnol.21:484-490, 2003. Methods of making domain antibodies comprising the variable domain of an antibody heavy chain or the variable domain of an antibody light chain, which contain three of the six naturally occurring HVRs or CDRs from the antibody, are also known in the art. See, for example, Muydermans,Rev. Mol. Biotechnol. 74:277-302, 2001。
in some embodiments, the anti-HER 2 antibody is a monoclonal antibody. As used herein, a monoclonal antibody refers to an antibody that is a substantially homogeneous antibody, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies are not a mixture of discrete antibodies. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or can be prepared by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. For example, monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al, 1990, Nature, 348: 552-.
In some embodiments, the anti-HER 2 antibody is a chimeric antibody. As used herein, a chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. A complete chimeric antibody comprises two copies of a chimeric light chain and two copies of a chimeric heavy chain. The generation of chimeric antibodies is known in the art (Cabilly et al (1984),Proc. Natl. Acad. Sci. USA,81:3273-3277, Harlow and Lane (1988),Antibodies: a Laboratory Manual,cold Spring Harbor Laboratory). Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic those of an antibody derived from one mammalian species, while the constant portions are homologous to sequences in an antibody derived from another mammalian species. One clear advantage of such chimeric forms is that, for example, the variable regions can be conveniently derived from currently known sources, using readily available hybridomas or B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. Although variable regions have the advantage of being easy to prepare, and specificity is not affected by their origin, human constant regions are less likely to elicit an immune response from a human subject when injected with an antibody than constant regions from non-human sources. However, the definition is not limited to this particular example.
In some embodiments, the anti-HER 2 antibody is a humanized antibody. As used herein, humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab ', F (ab') 2 Or other antigen binding subsequence of an antibody) that contains minimal sequences derived from non-human immunoglobulins. For the most partHumanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR or CDR of the recipient are replaced by residues from an HVR or CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody nor in the introduced HVRs or CDRs or framework sequences, but are included to further improve and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the HVRs or CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99/58572. Other forms of humanized antibodies have one or more (one, two, three, four, five, six) HVRs or CDRs that are altered relative to the original antibody, which are also referred to as "derived from" one or more HVRs or CDRs from the original antibody.
In some embodiments, the anti-HER 2 antibody is a human antibody. As used herein, a human antibody means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or has been prepared using any technique known in the art for preparing human antibodies. Human antibodies as used herein include antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses the human antibody (Vaughan et al, 1996, Nature Biotechnology, 14: 309-. Human antibodies can also be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). The process is described in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from an individual or can have been immunized in vitro). See, e.g., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), Boerner et al, 1991, J. Immunol, 147 (1) 86-95; and U.S. Pat. No. 5,750,373.
The anti-HER 2 antibodies described herein may further include analogs and derivatives that are modified (i.e., by covalently attaching any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity). For example, derivatives and analogs of antibodies include those that have been further modified (e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc.). Chemical modification can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formulation, and the like. In addition, the analog or derivative may contain one or more unnatural amino acid.
In some embodiments, amino acid sequence variants of the anti-HER 2 antibodies provided herein are contemplated. For example, it may be desirable to increase the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, the precursor being that the final construct has the desired characteristics, e.g., antigen binding. In some embodiments, anti-HER 2 antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs. Amino acid substitutions may be introduced into the antibody of interest and the product screened for desired activity, e.g., retaining/increasing antigen binding, reducing immunogenicity, or improving ADCC or CDC. Conservative substitutions are shown in table C below.
Table C: conservative substitutions
Original residue Exemplary substitutions Preferred substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln
Asp (D) Glu; Asn Glu
Cys (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn
Glu (E) Asp; Gln Asp
Gly (G) Ala Ala
His (H) Asn; Gln; Lys; Arg Arg
Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu
Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile
Lys (K) Arg; Gln; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr
Pro (P) Ala Ala
Ser (S) Thr Thr
Thr (T) Val; Ser Ser
Trp (W) Tyr; Phe Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu
Amino acids can be classified into different classes based on common side chain properties:
a. and (3) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
b. neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;
c. acidity: asp and Glu;
d. alkalinity: his, Lys, Arg;
e. residues that influence chain orientation: gly, Pro;
f. aromatic: trp, Tyr, Phe.
Non-conservative substitutions will require the replacement of one of these classes for another.
An exemplary substitution variant is parentAnd a force-matured antibody portion, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and variant antibody portions are displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g., substitutions) can be made in HVRs, for example, to improve antibody moiety affinity. Residues encoded by codons that undergo high frequency mutations during the maturation process of the somatic cells can be identified at HVR "hot spots" (see, e.g., Chowdhury,Methods Mol. Biol. 207:179-196 (2008)) and/or specificity-determining residues (SDR), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection from secondary libraries has been described in, for example, Hoogenboom et al,Methods in Molecular Biology 178:1-37 (O' Brien et al eds., Human Press, Totowa, NJ, (2001)).
Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of an antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.
In some embodiments, one or more amino acid modifications may be introduced into the Fc region of a full-length anti-HER 2 antibody provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variants have enhanced ADCC effector function, which is often associated with binding to an Fc receptor (FcR). In some embodiments, the Fc region variant has reduced ADCC effector function. There are several examples of changes or mutations in the Fc sequence that can alter effector function. For example, WO 00/42072 and Shields et alJ Biol. Chem. 6591-6604 (2001) describe antibody variants with improved or reduced binding to FcR. The contents of those publications are specifically incorporated herein by reference.
In some embodiments, the anti-HER 2 antibody comprises a monoclonal antibody having some but not allNot all effector functions of the Fc region, which makes it a desirable candidate for applications where the in vivo half-life of an anti-HER 2 antibody is important, while certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The primary cell NK cells used to mediate ADCC express only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in ravatch and Kinet,Annu. Rev. Immunol.9:457-492 (1991) in Table 3 on page 464. Non-limiting examples of in vitro assays to evaluate ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al)Proc. Nat'l Acad. Sci. USA7059 (1986)) and Hellstrom, I et al,Proc. Nat'l Acad. Sci. USA82:1499-,J. Exp. Med166: 1351-. Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry) TM Non-radioactive cytotoxicity assay (CellTechnology, inc. Mountain View, Calif.); and Cytotox 96 TM Non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be in vivo, e.g. in animal models such as Clynes et al Proc. Nat'l Acad. Sci. USA95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISAs. To evaluate complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al,J. Immunol. Methods202:163 (1996), Cragg, M, S, et al,Blood101:1045-,Blood103:2738-2743 (2004)). Can also use the powerFcRn binding and in vivo clearance/half-life assays are performed by methods known in the art (see, e.g., Petkova, s.b. et al,Int'l. Immunol. 18(12):1759-1769 (2006))。
antibodies with reduced effector function include antibodies in which one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region are substituted (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
Certain antibody variants with improved or reduced binding to FcR are described. (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; and Shields et al,J. Biol. Chem. 9(2): 6591-6604 (2001).)。
in some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al,J. Immunol164: 4178 (2000).
In some embodiments, the anti-HER 2 antibody comprises a variant Fc region comprising one or more amino acid substitutions that increase half-life and/or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005/0014934a1(Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826). For additional examples of Fc region variants, see also Duncan& Winter, Nature322:738-40 (1988), U.S. Pat. No. 5,648,260, U.S. Pat. No. 5,624,821, and WO 94/29351.
In some embodiments, the anti-HER 2 antibody is altered to increase or decrease the degree of glycosylation of the anti-HER 2 antibody. The addition or deletion of glycosylation sites of the anti-HER 2 antibody can be conveniently achieved by altering the amino acid sequence of the anti-HER 2 antibody or polypeptide portion thereof, such that one or more glycosylation sites are created or removed.
Where the anti-HER 2 antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched bi-antennary oligosaccharides, which are typically attached by an N-bond to Asn297 of the CH2 domain of the Fc region. See for example Wright et al,TIBTECH15:26-32(1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-HER 2 antibody of the invention may be modified to produce an anti-HER 2 antibody variant with certain improved properties.
The N-glycans attached to the CH2 domain of the Fc are heterogeneous. The antibody or Fc fusion protein produced in CHO cells is fucosylated by fucosyltransferase activity. See Shoji-Hosaka et al, j. biochem. 2006, 140: 777-83. Typically, a small fraction of naturally occurring afucosylated IgG is detectable in human serum. N-glycosylation of Fc is important for binding to Fc γ R; and afucosylation of the N-glycans increases the binding ability of Fc to Fc γ RIIIa. Increased Fc γ RIIIa binding can enhance ADCC, which can be advantageous in certain antibody therapeutic applications where cytotoxicity is desired.
Further provided are anti-HER 2 antibody variants having bisected oligosaccharides, e.g., wherein the biantennary oligosaccharides attached to the Fc region of the anti-HER 2 antibody are bisected by GlcNAc. Such anti-HER 2 antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al); U.S. Pat. No. 6,602,684 (Umana et al); US2005/0123546(Umana et al) and Ferrara et al,Biotechnology and Bioengineering, 93(5): 851-861 (2006). Also provided are oligosaccharides having at least one of the oligosaccharides attached to an Fc regionanti-HER 2 antibody variants of galactose residues. Such anti-HER 2 antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
In some embodiments, an anti-HER 2 antibody variant comprising an Fc region is capable of binding Fc γ RIII. In some embodiments, the anti-HER 2 antibody comprises a human wild-type IgG1 Fc region.
Also provided are one or more nucleic acids encoding the heavy and/or light chain of an anti-HER 2 antibody, vectors comprising the one or more nucleic acids, and methods of making the anti-HER 2 antibody.
Conjugation moieties
The conjugate moiety of the ADCs described herein comprise a toxin, such as a cytotoxic agent useful in cancer therapy. In some embodiments, the toxin is a chemotherapeutic agent. In some embodiments, the toxin is a small molecule drug. Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatins, CC-1065, duocarmycins, enediynes, geldanamycins, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof. In some embodiments, the conjugate moiety comprises Monodesulfonylcadaverine (MDC). In some embodiments, the conjugate moiety comprises TAM 1. In some embodiments, the conjugate moiety comprises monomethyl auristatin e (mmae).
Anthracyclines are derived from the bacterium streptomyces and have been used to treat a wide range of cancers such as leukemia, lymphoma, breast, uterine, ovarian and lung cancers. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., doxorubicin), epirubicin, idarubicin, valrubicin, and mitoxantrone.
Dolastatins and their peptide analogs and derivatives auristatins are highly potent antimitotic agents that have been shown to have anti-cancer and antifungal activity. See, for example, U.S. Pat. No. 5,663,149 and Pettit et al, Antimicrob. Agents Chemother. 42:2961-2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, auristatin E, auristatin eb (aeb), auristatin efp (aefp), MMAD, MMAF, MMAE, and 5-benzoylvaleric acid-AE Ester (AEVB).
Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92: 3642-. Exemplary dolastatins and auristatins include, but are not limited to (+) -duocarmycin A and (+) -duocarmycin SA, and (+) -CC-1065.
Enedialkynes are a class of anti-tumor bacterial products characterized by nine-and ten-membered rings or by the presence of a conjugated three-double-triple bond ring system. Exemplary enediynes include, but are not limited to, calicheamicin (calicheamicin), esperamicin (esperamicin), and dalensomycin (dynemicin).
Geldanamycin is a benzoquinone ansamycin antibiotic that binds Hsp90 (heat shock protein 90) and has been used as an antineoplastic agent. Exemplary geldanamycin species include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).
Maytansinoids or derivatives thereof maytansinoids inhibit cell proliferation by inhibiting microtubule formation during mitosis through the inhibition of tubulin polymerization. See Remilard et al, Science 189:1002-1005 (1975). Exemplary maytansinoids and maytansinoids include, but are not limited to mertansine (DM1) and its derivatives, and ansamitocins.
Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL) ® ) And docetaxel (TAXOTERE) ® )。
Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.
One skilled in the art can chemically modify the desired compound to render the reaction of the compound more convenient for the purpose of preparing the conjugates of the invention.
In some embodiments, the conjugate moiety comprises a toxin polypeptide (or toxin protein). Examples of toxin polypeptides include, but are not limited to, diphtheria toxin (diphtheria) a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain, ricin a chain, abrin a chain, gelonin a chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, pokeweed (phytolacca americana) proteins (PAPI, PAPII, and PAP-S), momordica charantia (momordia) inhibitors, leprosy proteins, croton toxins, saponaria officinalis (sapaonaria officinalis) inhibitors, gelonin, mitogellin, restrictocin, phenomycin (phenomycin), enomycin (enomycin), trichothecene toxins (tricocene), cystine Inhibitors (ICK) peptides (e.g., cex peptides) and conotoxins (e.g., KIIIA or iiia).
The conjugate moieties described herein contain an amine donor group conjugated to an acceptor glutamine residue in an anti-HER 2 antibody. Any amine donor group-free conjugate moiety may be indirectly conjugated to the anti-HER 2 antibody via a small molecule handle containing an amine donor group.
The term "amine donor group" as used herein refers to a reactive group containing one or more reactive amines (e.g., primary amines). For example, the conjugate moiety can comprise an amine donor group (e.g., primary amine-NH 2), a linker, and a toxin (e.g., a small molecule). The conjugate moiety can also be a polypeptide containing reactive Lys (e.g., endogenous Lys) or a biocompatible polymer. In some embodiments, the amine donor group is a primary amine (-NH2) that provides a substrate for transglutaminase to allow conjugation of the conjugate moiety to the anti-HER 2 antibody via the acceptor glutamine. Thus, the linkage between the donor glutamine and the amine donor group may be of the formula-CH 2 -CH 2 -CO-NH-.
In some embodiments, the anti-HER 2 antibody and the conjugate moiety are connected by a linker. The phrase "linker" refers to the attachment of one structural element of the compound to the same compoundStructural elements of the compound of (a) one or more other structural elements. In some embodiments, the linker is a non-cleavable linker. Suitable non-cleavable linkers include, but are not limited to, NH 2 -R-X、NH 2 NH-R-X and NH 2 -O-R-X, wherein R is an alkyl group or a polyethylene glycol group (also known as PEG), wherein X is a toxin. The polyethylene glycol group or PEG group may have the formula- (CH) 2 CH 2 O) n -, where n is an integer of at least 1. In some embodiments, n is any one of 2,4, 6, 8, 10, 12, 16, 20, or 24.
In some embodiments, the anti-HER 2 antibody and the conjugate moiety are connected by a cleavable linker. Suitable cleavable linkers include, but are not limited to, Lys-Phe-X, Lys-Val-Cit-PABC-X, NH 2 -(CH 2 CH 2 O) n -Val-Cit-PABC-X and NH 2 -(CH 2 CH 2 O) n -(Val-Cit-PABC-X) 2 Wherein X is a toxin and n is an integer of at least 1 (such as any of 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24). PABC refers to p-aminobenzyloxycarbonyl. Cit refers to citrulline.
Other exemplary amine donor group-linkers include, but are not limited to, Ac-Lys-Gly, aminocaproic acid, Ac-Lys- β -Ala, amino-PEG 2 (polyethylene glycol) -C2, amino-PEG 3-C2, amino-PEG 6-C2, Ac-Lys-Val (valine) -Cit (citrulline) -PABC (p-aminobenzyloxycarbonyl), aminocaproyl-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.
In some embodiments, the conjugate moiety is via-NH- (C) n The linker is linked to the acceptor glutamine residue, wherein (C) n Is a substituted or unsubstituted alkyl or heteroalkyl chain wherein n is an integer from about 1 to about 60. In some embodiments, the carbon of the chain is substituted with alkoxy, hydroxy, alkylcarbonyloxy, alkyl-S-, thiol (thio), alkyl-c (o) S-, amine, alkylamine, amide or alkylamide. In some embodiments, n is from about 2 to about 20.
In some embodiments, the linker is branched. In some embodiments, the linker is linear. In some embodiments, the linker has more than one (such as 2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) attachment site for attaching an active moiety. These active moieties may be the same or different from each other. For example, the conjugate moiety may comprise a polyacetal-based or polyacetal derivative-based polymer linked to a plurality of toxins (such as chemotherapeutic agents).
In some embodiments, the conjugate moiety comprises a toxin selected from the group consisting of: Ac-Lys-Gly-MMAD, amino-PEG 3-C2-MMAD, amino-PEG 6-C2-MMAD, amino-PEG 3-C2-amino-nonanoyl-MMAD ], aminocaproyl-Val-Cit-PABC-MMAD, Ac-Lys-beta-Ala-MMAD, aminocaproyl-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl-MMAE, amino-PEG 3-C2-MMAE, amino-PEG 2-C2-MMAE, aminocaproyl-MMAF, aminocaproyl-Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, amino-MM 2-C2-PEG, amino-PEG 3-C2-MMAF, Putrescine-geldanamycin and Ac-Lys-putrescine-geldanamycin. In some embodiments, the amine donor agent is aminocaproyl-Val-Cit-PABC-MMAE, aminocaproyl-Val-Cit-PABC-MMAF, Ac-Lys-putaminyl-geldanamycin, Ac-Lys- β -Ala-MMAD, Ac-Lys-Val-Cit-PABC-MMAD, aminocaproyl-Val-Cit-PABC-MMAD, and amino-PEG 6-C2-MMAD.
In some embodiments, the conjugate moiety is a maytansine derivative. In some embodiments, the conjugate moiety is a non-cleavable linker (such as amino- (CH) 2 CH 2 O) n Linker) is used. In some embodiments, the conjugate moiety has the chemical structure of formula (II):
Figure 394260DEST_PATH_IMAGE011
(II),
wherein n is an integer selected from 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11 and 12.
In some embodiments, the conjugate moiety is a peptide comprising a cleavable linkerMMAE derivatives of (such as amino- (CH) 2 CH 2 O) n -Val-Cit-PABC-MMAE). In some embodiments, the conjugate moiety has the chemical structure of formula (III):
Figure 226212DEST_PATH_IMAGE012
wherein n is an integer selected from 1, 2, 3,4, 5,6, 7, 8, 9, 10, 11 and 12.
In some embodiments, the conjugate moiety comprises a toxin having the chemical structure of formula (I):
Figure 484018DEST_PATH_IMAGE013
in some embodiments, the toxin is LND 1002. LND1002 is a MMAE-derived toxin having a PEG linker with a primary amine group for conjugation. LND1002 has the chemical structure of formula (I).
Chemical compounds contemplated herein include salts, solvates, or stereoisomers thereof, including all permutations of salts, solvates, and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compounds.
The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of compounds derived from various organic and inorganic counterions well known in the art and including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.
The term "salt thereof" means a compound formed when the proton of an acid is replaced by a cation, such as a metal cation or an organic cation, and the like. Where applicable, the salts are pharmaceutically acceptable salts, although this is not required for salts of intermediate compounds which are not intended for administration to a patient. By way of example, salts of the compounds of the present invention include those in which the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
"solvate" refers to a complex formed by a solvent molecule in combination with a molecule or ion of a solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, ethanol, isopropanol, and mixtures thereof,N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide and water. When the solvent is water, the solvate formed is a hydrate.
"one stereoisomer" and "stereoisomers" refer to compounds having the same atomic connectivity but differing in atomic arrangement in space. Stereoisomers include cis-trans isomers,EAndZisomers, enantiomers, and diastereomers.
Method for preparing antibody-drug conjugates
The ADCs described herein may be prepared using any suitable method in the art (e.g., by conjugating an amine group in a conjugate moiety to an endogenous acceptor glutamine residue in an antibody using transglutaminase). See, for example, U.S. patent No. 10,357,472, which is incorporated by reference herein in its entirety.
In some embodiments, the anti-HER 2 ADC is prepared using a wild-type or engineered transglutaminase. Engineered transglutaminases suitable for use in preparing the ADCs described herein include those described in U.S. patent No. 10,471,037 (which is incorporated herein by reference in its entirety).
TGase is produced by the addition of lysine donor residues in one protein and acceptor glutamine residues in another proteinProtease resistant isopeptide bonds are formed between groups to catalyze covalent protein cross-linking with concomitant release of ammonia. The catalytic mechanism of transglutaminase has been proposed as follows. After binding of the enzyme to the first substrate (receptor or Q-substrate) containing glutamine, it forms a γ -glutamyl thioester, called acylase intermediate, with release of ammonia, with a cysteine residue in the active center of TGase. The second substrate (donor or K-substrate) then binds to the acylase intermediate and attacks the thioester bond. The product (two proteins cross-linked by N epsilon (gamma-glutamyl) lysine-isopeptide bridge) is formed and released. This re-establishes the active central Cys residue of the enzyme in its original form and allows it to participate in another catalytic cycle. The formation of the covalent acylase intermediate is believed to be the rate limiting step in these reactions. Many catalytic triads of transglutaminase are papain-like, containing a Cys-His-Asp (where His is histidine and Asp is aspartic acid) and a tryptophan (Trp) residue, critically located 36 residues away from the active center Cys. In contrast, from streptoverticillium species (Streptoverticillium sp) The isolated bacterial TGase (see above) has an atypical catalytic triad and shows no sequence homology with the papain-like catalytic triads of other tgases.
Several types of transglutaminase have been reported in various living organisms, including microbial organisms. Examples are TGase from guinea pig liver (GTGase), fish liver (FTGase) and microorganisms (mTGase) and any recombinant TGase (rtgase). Other tgases than those listed herein may also be used according to the present invention. Examples of useful tgases include microbial transglutaminase, such as, for example, streptomyces mobaraensis (r) as disclosed in U.S. Pat. No. 5,156,956Streptomyces mobaraense) Streptomyces cinnamomi (A)Streptomyces cinnamoneum) And Streptomyces griseofuscus: (Streptomyces griseocarneum) And Streptomyces lavendulae (S.lavendulae) disclosed in U.S. Pat. No. 5,252,469Streptomyces lavendulae) And Streptomyces ladakaensis disclosed in JP2003199569 (a)Streptomyces ladakanum). Other useful microbial transglutaminases have been obtained from Bacillus subtilis (disclosed in U.S. Pat. No. 5,731,183) and eachSeparating the seed slime mold. A further example of a useful microbial transglutaminase is WO 96/06931 (e.g.fromBacilus lydicusTransglutaminase according to (3) and those disclosed in WO 96/22366. Useful non-microbial transglutaminase includes transglutaminases from the liver of guinea pigs and from various marine sources such as flat fish Pagrus major (disclosed in EP-0555649) and Crassostrea gigas (Crassostrea gigas)Crassostrea gigas) Transglutaminase (disclosed in U.S. Pat. No. 5,736,356). An exemplary TGase is Bacterial Transglutaminase (BTG) (see e.g., EC 2.3.2.13, protein-glutamine- γ -glutamyltransferase). In another exemplary embodiment, the TGase is from Streptomyces mobaraensis (C.)S. mobaraense). In another embodiment, the TGase is a mutant (e.g., engineered) TGase having at least 80% sequence homology to a native TGase. An example is recombinant bacterial transglutaminase from streptomyces mobaraensis (available from Zedira, Darmstadt, Germany).
Streptomyces ladakaensis: (Streptomyces ladakanum) ATCC 27441 or NRRL3191 mTgase is expressed as pre-mTGase pro (GenBank accession number AY 241675). There are 410 amino acid residues in the pre-mTGase pro-enzyme, 331 amino acids in the mature enzyme plus 30 amino acids of the precursor (pre) and 49 amino acids of the proenzyme (pro). The propeptides are strong inhibitors of the maturase enzyme. Primers designed according to AY241675 were used to clone both mTgase pro-and mature mTgase from ATCC 27441DNA into the Nde I and Xho I sites of pET29b (+) vector. Active mTgase can be obtained from enterokinase light chain (EKL) digestion of pro-mTgase or refolding of mature mTgase. From Streptomyces ladakaensis (C.), (C.Strep Ladakanum) mTgase (TG _ SL) of (D) is very similar to that from Streptomyces mobaraensis: (A)Strep. mobaraensis) mTgase (TG _ SM, marketed by Ajinomoto as ACTIVA) with a small number of amino acid differences.
The transglutaminase used in the methods described herein can be obtained or prepared from a variety of sources. In some embodiments, the transglutaminase is a calcium-dependent transglutaminase requiring calcium to induce a conformational change in the enzyme and allow enzymatic activity. For example, transglutaminase can be derived from guinea pig liver and obtained from commercial sources (e.g., Sigma-A)ldrich (St Louis, Mo.) and MP Biomedicals (Irvine, Calif.)). In some embodiments, the transglutaminase is a calcium-independent transglutaminase that does not require calcium to induce a conformational change in the enzyme and allows enzymatic activity. In some embodiments, the transglutaminase is a microbial transglutaminase derived from a microbial genome, such as from streptoverticillium or streptomyces (bStreptomices) (e.g., Streptomyces mobaraensis or streptoverticillium mobaraense: (Streptoverticillium mobarensis) Transglutaminase of (c). In some embodiments, the transglutaminase is a mammalian protein (e.g., human transglutaminase), a bacterial protein, a plant protein, a fungal protein (e.g., oomycetes: (i.e., escherichia coli)Oomycetes) And actinomycetes (A), (B), (C)actinomycetes) Transglutaminase) or prokaryotic proteins. In some embodiments, the transglutaminase is from the genus Micrococcus (Micrococcus: (Micrococcus)Micrococcus) Clostridium (f) <Clostridium)、TurolpsisRhizopus genus (A), (B), (C), (B), (C)Rhizopus) Monascus genus (Monascus) Or of the genus Bacillus (Bacillus)。
In some embodiments, the transglutaminase used in the methods described herein is a recombinant protein produced using recombinant techniques. In some embodiments, the TGase is prepared by: (a) culturing a host cell (such as a prokaryotic cell) comprising a vector comprising a nucleic acid encoding a zymogen of a TGase, and (b) obtaining a mature TGase by cleavage of the pro sequence of the zymogen (e.g., by an endo kinase (endokinase) light chain). In some embodiments, the TGase is purified by chromatography (such as by affinity chromatography or ion exchange chromatography). In some embodiments, the TGase is labeled (such as his-label) to facilitate purification.
In some embodiments, the anti-HER 2 ADC is prepared by contacting an anti-HER 2 antibody with a conjugate moiety in the presence of transglutaminase under conditions sufficient to produce the ADC, wherein the anti-HER 2 antibody comprises an N-glycosylated Fc region, wherein the N-glycosylated Fc region comprises acceptor glutamine residues flanking an N-glycosylation site, and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residues. In some embodiments, the anti-HER 2 ADC is prepared by contacting a composition comprising an anti-HER 2 antibody with a conjugate moiety in the presence of transglutaminase under conditions sufficient to produce the ADC, wherein at least some (e.g., at least about 50%, 60%, 70%, 80%, 90% or more) of the anti-HER 2 antibody comprises an N-glycosylated Fc region, wherein the Fc region comprises acceptor glutamine residues flanking an N-glycosylation site, and wherein the conjugate moiety is conjugated to the anti-HER 2 antibody via the acceptor glutamine residues.
In some embodiments, the anti-HER 2 ADC is prepared in two steps. First, the small molecule handle was conjugated to an anti-HER 2 antibody via TGase to generate an intermediate conjugate. Subsequently, the toxin is coupled, covalently or non-covalently, to the intermediate conjugate via a small molecule handle. Small molecule handles can be specifically designed to tailor the conjugation of toxins, thus allowing conjugation of any kind of toxin to the anti-HER 2 antibody. The two-step process is particularly useful when the supply of anti-HER 2 antibody and/or toxin is limited, and when the toxin has low water solubility and/or induces aggregation of anti-HER 2 antibody.
The small molecule handles described herein typically have an-NH-group 2 -the structure of R, wherein R is a moiety allowing attachment of a toxin. The introduction of a small molecule handle in the methods described herein significantly increases the flexibility of the methods. In particular, the structure of the small molecule handle can be tailored to attach a desired toxin. For example, in some embodiments, R is a ligand that specifically binds to a binding partner. This allows for the attachment of any molecule (such as a protein) containing a binding partner. Suitable ligand/binding partner pairs include, but are not limited to, antibodies/antigens, antigens/antibodies, avidin/biotin, biotin/avidin, streptavidin/biotin, biotin/streptavidin, glutathione/GST, GST/glutathione, maltose binding protein/amylose, amylose/maltose binding protein, cellulose binding protein and cellulose, cellulose/cellulose binding protein, and the like.
Other suitable small molecule handles described herein include, but are not limited to, NH 2 -CH 2 -CH(OH)-CH 2 -NH 2 、NH 2 -R-(OR') 2 、NH 2 -R=O、NH 2 -R-SH、NH 2 -R-azide. These small molecule handles allow for the passage of suitable linkers such as NH 2 -O-R-X, maleimide-R-X and cyclooctyne-R- (R' -X) 2 Attaching a conjugate moiety, wherein X is an active moiety and R' are independently a linker group, such as a linker group comprising an alkyl group or a polyethylene glycol group.
The reaction catalyzed by TGase may be carried out for several hours to one day (e.g. overnight). The conjugate moiety or small molecule handle is reacted with an anti-HER 2 antibody (e.g., 1 mg/mL) at a ligand concentration of 400 to 600 μmol/L, which provides a 60-90 fold excess of substrate compared to the anti-HER 2 antibody, or at a lower excess of substrate, e.g., 1 to 20 fold, or 10-20 fold. The reaction can be carried out in potassium-free phosphate buffered saline (PBS; pH 8) at 37 ℃. After 4 hours to several days, steady state conditions were reached. Excess ligand and enzyme were then removed using centrifugation-dialysis (VIVASPIN MWCO 50kDa, Vivascience, Winkel, Switzerland) or diafiltration (PELLICON MWMCO 50kDa, Millipore). The reaction can be monitored by HPLC.
The resulting ADC may be analyzed using any suitable method. For example, the stoichiometry of the ADC can be characterized by liquid chromatography-mass spectrometry (LC/MS) using the top-down method to assess the number of conjugate moieties conjugated to the antibody, particularly the homogeneity of the composition. The conjugate can be reduced prior to LC/MS analysis and the light and heavy chains measured separately.
In one embodiment, the drug loading of the ADC (e.g., the number of toxins in each conjugate of the anti-HER 2 antibody) is analyzed. Such methods can be used to determine the average number of conjugate moieties or toxins (such as MMAE) per anti-HER 2 antibody and the distribution of the number of conjugate moieties or toxins (such as MMAE) per antibody in the composition, i.e., the percentage of total antibody with any given level of drug loading or DAR. Antibody moieties having a number (n) of conjugated acceptor glutamines (e.g., n =1, 2, 3,4, 5,6, etc.) can be determined. One technique suitable for such assays and more generally for drug loading is Hydrophobic Interaction Chromatography (HIC), which may be as described for example for Hamblett et al (2004) Cancer res.10: 7063-; wakankar et al (2011) mAbs 3(2) 161-172; and Lyon et al (2012) Methods in Enzymology, Vol. 502:123-138, the disclosure of which is incorporated herein by reference.
The molar ratio between transglutaminase and anti-HER 2 antibody in the conjugation reaction can be controlled to allow for an efficient transglutaminase reaction. For example, in some embodiments, the molar ratio of the transglutaminase and the anti-HER 2 antibody is about 10:1 to about 1: 100. The amount of transglutaminase in the reaction mixture may be controlled to allow for an efficient transglutaminase reaction. For example, in some embodiments, the concentration of transglutaminase in the reaction mixture is any of about 0.01 mg/ml to about 5 mg/ml. In some embodiments, the concentration ratio between the conjugate moiety and the anti-HER 2 antibody is from about 2:1 to about 800: 1. In some embodiments, the conjugation efficiency of the anti-HER 2 antibody and conjugate moiety is at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 90% or more). Conjugation efficiency can also be measured at different temperatures (such as room temperature or 37 ℃).
In some embodiments, the ADC is purified after the conjugation reaction. For example, the ADC may be purified using affinity chromatography (such as a protein a column and/or a size exclusion column).
Pharmaceutical compositions, kits and articles of manufacture
Also provided are pharmaceutical compositions comprising any of the antibody-drug conjugates described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises about 50mM sodium citrate, about 10 mM citric acid, about 4.0% (w/v) sucrose, and about 0.02% (w/v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 10mg/mL of ADC.
The term "pharmaceutically acceptable carrier" is used herein to describe any ingredient other than a compound of the invention. The choice of excipient depends to a large extent on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In some embodiments, isotonic agents, including, but not limited to, sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, are included in the pharmaceutical composition. Additional examples of pharmaceutically acceptable substances include, but are not limited to, wetting or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf-life or effectiveness of the antibody.
In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, a pH range of any of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition may also be made isotonic with blood by the addition of a suitable tonicity modifier, such as glycerin.
Pharmaceutical compositions to be used for in vivo administration are typically formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration. Sterility is readily achieved by filtration through sterile filtration membranes. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition may be in the form of a liquid solution, for example in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, the pharmaceutical compositions may be in solid form and re-dissolved or suspended immediately prior to use. Lyophilized compositions are also included.
In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is contained in a disposable vial, such as a disposable sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.
The pharmaceutical compositions described herein may be prepared, packaged or sold in bulk as a single unit dose or as multiple single unit doses. As used herein, a "unit dose" is an individual amount (discrete amount) of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is typically equal to the dose of active ingredient to be administered to the subject or a convenient fraction of such dose, e.g., half or one third of such dose.
In some embodiments, the pharmaceutical compositions described herein are suitable for parenteral administration. Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical disruption of the tissue of the subject and administration of the pharmaceutical composition through a gap in the tissue, thus generally resulting in direct administration into the blood stream, muscle or internal organ. For example, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue penetrating non-surgical wound, and the like. In particular, it is contemplated that parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intrasynovial injection or infusion; and renal dialysis infusion techniques. In some embodiments, the pharmaceutical composition is suitable for intravenous administration.
Formulations of pharmaceutical compositions suitable for parenteral administration may be prepared, packaged or sold in a form suitable for bulk administration or continuous administration. Injectable preparations may be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably to a pH of 3-9), but, for some applications, they may more suitably be formulated as sterile non-aqueous solutions or used in combination with a suitable vehicle, such as sterile, pyrogen-free water, in dry form. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered if desired. Other parenterally administrable formulations that may be used include those comprising the active ingredient in microcrystalline form or in liposomal preparations. Formulations for parenteral administration may be formulated for immediate release and/or engineered release. Engineered release formulations include controlled, delayed, sustained, pulsed, targeted, and programmed release formulations. For example, in one aspect, sterile injectable solutions can be prepared by incorporating the required amount of ADC in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
The dosage regimen may be adjusted to provide the best desired response. For example, a single large dose may be administered, or several separate doses may be administered over time. Parenteral compositions are particularly advantageously formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the patients/subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
The present application also provides kits (or articles of manufacture) for use in the treatment of cancer as described herein. The kit may comprise one or more containers comprising any of the ADCs for use in the treatment of cancer (e.g., HER 2-positive cancer). The kits described herein may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts, as well as instructions for performing any of the methods described herein. In some embodiments, the kit comprises instructions for administering the ADC to treat cancer, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, or lung cancer. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether the individual has cancer and a stage of cancer. Instructions relating to the use of the ADC typically include information about the dosage, dosing regimen and route of administration for the intended treatment. The container may be a unit dose, a large package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc) are also acceptable.
The kit is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging is also contemplated for use in combination with a particular device, such as an infusion device, e.g., a micro-pump. The kit may have a sterile access port, for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The container may further comprise a second pharmaceutically active agent. These articles may be further sterilized and/or sealed.
Exemplary embodiments
Embodiments provided herein are:
1. a method of treating HER 2-positive cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous acceptor glutamine residue, and wherein the conjugate moiety is conjugated to the acceptor glutamine residue.
2. The method of embodiment 1, wherein said HER 2-positive cancer is HER 23 + as determined by an Immunohistochemistry (IHC) test.
3. The method of embodiment 1, wherein the HER 2-positive cancer is HER 22 + as determined by an IHC test.
4. The method of embodiment 1, wherein the HER 2-positive cancer is positive as determined by a Fluorescence In Situ Hybridization (FISH) test.
5. The method of any one of embodiments 1-4, wherein the individual is non-responsive or not eligible for standard therapy.
6. The method of embodiment 5, wherein said individual has not previously received a second HER2-targeting agent.
7. The method of any one of embodiments 1-4, wherein the individual has previously received a second HER2-targeting agent.
8. The method of embodiment 7, wherein said HER 2-positive cancer is resistant or refractory to a second HER2-targeting agent.
9. The method of embodiment 7 or 8, wherein the second HER2 targeting agent is trastuzumab, emrituximab, pertuzumab, or lapatinib.
10. The method of any one of embodiments 1-9, wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg.
11. A method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, wherein the conjugate moiety is conjugated to the receptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg.
12. The method of embodiment 11, wherein said cancer is a solid cancer.
13. The method of embodiment 12, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
14. The method of any one of embodiments 11-13, wherein the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg.
15. The method of any one of embodiments 11-13, wherein the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg to about 8 mg/kg.
16. The method of embodiment 15, wherein the antibody-drug conjugate is administered at a dose of about 0.3 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.5 mg/kg, about 6.0mg/kg, or 8.0 mg/kg.
17. The method of any one of embodiments 1-16, wherein the antibody-drug conjugate is administered intravenously.
18. The method of any one of embodiments 1-17, wherein the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once a week.
19. The method of any one of embodiments 1-18, wherein the individual is a human.
20. The method of any one of embodiments 1-19, wherein the Fc region of said anti-HER 2 antibody is N-glycosylated.
21. The method of any one of embodiments 1-20, wherein the receptor glutamine residue is Q295 in the heavy chain of the anti-HER 2 antibody according to EU numbering.
22. The method of any one of embodiments 1 to 21, wherein each heavy chain of the HER2 antibody is conjugated to a conjugate moiety.
23. The method of any of embodiments 1-22, wherein the conjugation moiety is conjugated to the acceptor glutamine residue via transglutaminase.
24. The method of any one of embodiments 1-23, wherein said anti-HER 2 antibody comprises: a heavy chain variable region (VH) comprising: heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and a light chain variable region (VL) comprising: light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO. 6.
25. The method of embodiment 24, wherein said anti-HER 2 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO. 7 and a VL comprising the amino acid sequence of SEQ ID NO. 8.
26. The method of any one of embodiments 1-25, wherein the Fc region is IgGl Fc.
27. The method of embodiment 26, wherein said anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10.
28. The method of any one of embodiments 1-27, wherein said toxin is monomethyl auristatin e (mmae).
29. The method according to any one of embodiments 1 to 28, wherein the conjugate moiety comprises a cleavable linker.
30. The method of embodiment 29, wherein the conjugate moiety is a compound of formula I:
Figure 382704DEST_PATH_IMAGE014
31. the method of any one of embodiments 1-30, wherein the antibody-drug conjugate is DP303 c.
Examples
The invention may be further understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.
Example 1 preparation and formulation of DP303 c.
DP303c is an antibody drug conjugate designed to target epidermal growth factor receptor 2 (HER2) positive cancers in humans. More specifically, the product is a monoclonal IgG1 antibody (DP001) targeting HER2 site-specifically conjugated with a cleavable LND1002 (toxin) at glutamine 295 in the constant region of each heavy chain. DP303c has a DAR (drug-to-antibody ratio) of about 2.0, such as 1.8 to 2.2.
DP001 is an anti-HER 2 monoclonal antibody having affinity with trastuzumab (HERCEPTIN) ® ) The same amino acid sequence. Specifically, it contains 1328 amino acids with two Heavy Chains (HC) of 450 amino acids (49284.65 Da, SEQ ID NO:9) and two Light Chains (LC) of 214 amino acids (23443.10 Da, SEQ ID NO: 10). DP001 is a heterotetramer of two HC subclasses of IgG1 and two LC subclasses of κ subclass linked by 16 disulfide bonds (12 intra-and 4 inter-chain). A schematic structure of DP001 is depicted in fig. 1.
LND1002 is a toxin derived from MMAE with a PEG linker and a primary amine group for conjugation to antibodies. LND1002 has the chemical structure of formula (I) as shown below:
Figure 483384DEST_PATH_IMAGE015
DP303c was obtained by conjugating DP001 to LND1002, which conjugation is catalyzed by microbial transglutaminase (mTgase). Briefly, DP001, reaction buffer pH 8.0 (50 mM Tris-acetate, 2mM EDTA, 0.1% Tween-20), and LND1002 in DMSO were loaded into sterile, flexible, ethylene-vinyl acetate bags under a laminar flow hood in a clean room. Once the mixture reached the desired temperature, mTGase was added to the bag by sterile syringe to start the conjugation reaction. The bags were then incubated at 30 ℃ for up to 120 hours. The reaction was monitored using RP-HPLC. The conjugation reaction was considered complete when the heavy chain conversion reached > 95%.
Using sterilized AKTA ® Ready System, the completed reactants were loaded in 30-35g DP303c/L resin into sterilized CAPTIVA ® The minimum residence time was 5 minutes on the protein a column to ensure complete binding of the DP303c product. Using an on-line 0.2 μm depth filter, any particles were removed from the reaction and/or buffer during purification, andkeeping the sterility. The column is then washed with excess binding and washing buffer to remove mTGase, unreacted LND1002 and any unwanted buffer components prior to low pH elution of the desired product. The eluted fractions were collected in a sterile collection bag pre-filled with a neutralization buffer.
The purified DP303c was formulated as follows: 50mM sodium citrate, 10 mM citric acid, 4.0% (w/v) sucrose and 0.02% (w/v) 10mg/mLDP303c in polysorbate 20. The concentrated components of the DP303c formulation were prepared in sterile containers and filtered through a 0.2 μm sterile PES filter and added to the DP303c concentrated stock solution. The formulation step is performed in a laminar flow hood in a clean room. The finally prepared solution was then filtered through a 0.2 μm sterile PES filter. The final filtered product was stored at-20 ℃ for long term storage.
Example 2 cytotoxic Effect of DP303c against HER 2-highly expressed cell lines compared to DP001 and T-DM1
Evaluation of DP303c, DP001 and T-DM1 (also known as Enmetuzumab (KADCYLA) in a cell-based assay using HER 23 + human breast cancer SK-BR-3 cell line ® ) Cytotoxicity (Li JY et al, Cancer Cell 29, 117-129).
SK-BR-3 cells were grown to about 70% confluence in tissue culture flasks, and then the medium was aspirated from the flasks and the cells were washed with small amounts of DPBS without magnesium and calcium. Cells were trypsinized to lift adherent cells and resuspended in fresh medium to approximately 1X 10 5 Final concentration of individual cells/mL. The resuspended cells were transferred to wells of a 96-well plate and incubated overnight at 37 ℃, 5% CO2, 85% relative humidity to allow the cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 was added to the wells at a final concentration of 500 ng/mL, 167 ng/mL, 55.6 ng/mL, 19 ng/mL, 6.2 ng/mL, 2.06 ng/mL, 0.69 ng/mL, or 0.229 ng/mL. DP001, DP303c or T-DM1 were added in triplicate at each concentration. Fresh medium was used as a negative control. The plates were then gently shaken on a plate shaker for 15-20 seconds and incubated at 85% humidity for 3 days.
After incubation, resazurin (Sigma, catalog No. 199303, batch No. MKBP3801V) was added to eachWells to a final concentration of about 0.0005% o and gently mixed with a plate shaker. The plates were further incubated for 3 hours. Subsequently, at SPECTRAMAX GEMINIXS ® On the plate reader, at λ ex 555 / λ em 585nm (570nm cutoff), and the plate was assayed using the template of the cell-based assay protocol. The mean RFU of the blank media wells was subtracted from the RFU readings of all wells with DP001, DP303c or T-DM1 treated cells. Software XLFit Excel version (4.3.2 Build11) (ID Business Solutions Limited) fitting model 201 was used to calculate IC 50 . Table 1 below compares the IC of DP001, DP303c and T-DM1 in SK-BR-3 cells 50 。IC 50 Indicating a higher cytotoxic effect of DP303c and T-DM1 compared to DP 001.
TABLE 1
IC 50 (ng/mL) IC 50 (nM)
DP001 175.2 1.19
DP303c 19.2 0.131
T-DM-1 27.4 0.189
FIG. 2 shows cell proliferation inhibition curves of SK-BR-3 cells after treatment with DP001 (blue circles and blue lines), DP303c (green triangles and green lines) or T-DM1 (brown squares and brown lines). As shown in FIG. 2, DP001, DP303c and T-DM1 were cytotoxic against SK-BR-3 cells (a HER2 high expressing cell line). DP303c has similar cytotoxic effects against this HER2 high expressing cell line as T-DM 1. Both DP303c and T-DM1 killed SK-BR-3 cells more strongly than DP001 (a HER2 targeting antibody).
Example 3: cytotoxic Effect of DP303c against HER2 Low expressing cell lines compared to DP001 and T-DM1
Cytotoxicity of DP303c, DP001 and T-DM1 was assessed in a Cell-based assay using the HER 22 + breast Cancer JIMT-1 Cell line (Li JY et al, Cancer Cell 29,117- & 129).
JIMT-1 cells were grown to about 70% confluence in tissue culture flasks, and then the medium was aspirated from the flasks and the cells were washed with small amounts of DPBS that did not contain magnesium and calcium. Cells were trypsinized to lift adherent cells and resuspended in fresh medium to approximately 1X 10 5 Final concentration of individual cells/mL. The resuspended cells were transferred to wells of a 96-well plate and incubated overnight at 37 ℃, 5% CO2, 85% relative humidity to allow the cells to adhere. After overnight incubation, DP001, DP303c or T-DM1 was added to each well at a final concentration of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL or 2.44 ng/mL. Each concentration of DP001, DP303c or T-DM1 was added in triplicate. Fresh medium was used as a negative control. The plates were then gently shaken on a plate shaker for 15-20 seconds and incubated at 85% humidity for 5 days.
After incubation, resazurin (Sigma, catalog No. 199303, lot No. MKBP3801V) was added to each well to a final concentration of about 0.0005 ‰, and gently mixed with a plate shaker. The plates were further incubated for 3 hours. Subsequently, at SPECTRAMAX GEMINIXS ® On the plate reader, at λ ex 555 / λ em 585nm (570nm cutoff), and the plate was assayed using the template of the cell-based assay protocol. All from cells treated with DP001, DP303c or T-DM1The mean RFU of the blank medium wells was subtracted from the RFU readings of the wells. Software XLFit Excel version (4.3.2 Build11) (ID Business Solutions Limited) fitting model 201 was used to calculate IC 50 . Table 2 below compares the IC of DP001, DP303c and T-DM1 in JIMT-1 cells 50 。IC 50 It was shown that DP303c has a higher cytotoxic effect than T-DM 1.
TABLE 2
IC 50 (ng/mL) IC 50 (nM)
DP001 N/A N/A
DP303c 62.6 0.42
T-DM-1 1282.3 8.66
FIG. 3 is a graph showing the cell proliferation inhibition curves of JIMT-1 cells after treatment with DP001 (blue circles and blue lines), DP303c (green triangles and green lines), or T-DM1 (brown squares and brown lines). As shown in FIG. 3, both DP303c and T-DM1 were cytotoxic against the JIMT-1 cell line, a HER2 low-expressing cell line. DP303c was more cytotoxic against this HER2 low expressing cell line than TDM-1.
Example 4: cytotoxic Effect of DP303c against HER2 negative cell line compared to DP001 and T-DM1
Cytotoxicity of DP303c, DP001 and T-DM1 against Hs746T cells was evaluated in cell-based assays. Hs746T is a human gastric Cancer cell line that expresses little to no HER2 (Corso S, et al, Mol Cancer 2010; 9: 121).
Hs746T cells were grown to about 70% confluence in tissue culture flasks, and then the medium was aspirated from the flasks and the cells were washed with small amounts of DPBS without magnesium and calcium. Cells were trypsinized to lift adherent cells and resuspended in fresh medium to approximately 1X 10 5 Final concentration of individual cells/mL. The resuspended cells were transferred to wells of a 96-well plate and incubated overnight at 37 ℃, 5% CO2, 85% relative humidity to allow the cells to adhere. After overnight incubation, DP001, DP303c, or T-DM1 was added to each well at a final concentration of 10,000 ng/mL, 2,500 ng/mL, 625 ng/mL, 156 ng/mL, 39.1 ng/mL, 9.77 ng/mL, 2.44 ng/mL, or 0.610 ng/mL. Each concentration of DP001, DP303c or T-DM1 was added in triplicate. Fresh medium was used as a negative control. The plates were then gently shaken on a plate shaker for 15-20 seconds and incubated at 85% humidity for 5 days.
After incubation, resazurin (Sigma, catalog No. 199303, lot No. MKBP3801V) was added to each well to a final concentration of about 0.0005% o and gently mixed with a plate shaker. The plates were further incubated for 3 hours. Subsequently, at SPECTRAMAX GEMINIXS ® On the plate reader, at λ ex 555 / λ em 585nm (570nm cutoff), and the plate was assayed using the template of the cell-based assay protocol. The mean RFU of the blank media wells was subtracted from the RFU readings of all wells with DP001, DP303c or T-DM1 treated cells. Software XLFit Excel version (4.3.2 Build11) (ID Business Solutions Limited) fitting model 201 was used to calculate IC 50
FIG. 4 shows the inhibition of cell proliferation of Hs746T cells treated with DP001 (blue circles and blue lines), DP303c (green triangles and green lines), or T-DM1 (brown squares and brown lines). As shown in FIG. 4, DP001, T-DM1, and DP303c did not show cytotoxic effects against Hs746T cells (a HER2 negative cell line).
Example 5: antitumor Effect of DP303c on mouse xenograft NCI-N87 cancer model compared to T-DM1
The in vivo antitumor activity of DP303c was studied in the NCI-N87 mouse xenograft model. NCI-N87 is a gastric cancer cell line that overexpresses HER2 (HER 23 +). NCI-N87 cells were implanted subcutaneously into female athymic nude mice. Tumor-bearing mice were randomly divided into 7 treatment groups (6 mice/group) and were treated by a single intravenous injection of either blank, DP303c or T-DM 1.
Relative Tumor Volume (RTV) was calculated according to the following formula: RTV = TV n /TV 0 Wherein TV n Is the tumor volume at day n, and TV 0 Is the tumor volume at day 0. Tumor growth inhibition ratio (TGI) was calculated using the following formula: TGI (%) = (1-T/C) × 100, where T/C is determined by calculating T/C = (average RTV of treatment group)/(average RTV of control group).
DP303c was effective in inhibiting tumor growth in the NCI-N87 xenograft model (FIG. 5). Single dose treatment of tumor-bearing mice with 4 or 8 mg/kg DP303c resulted in tumor regression (TGI of 81.1% and 92.5%) with no regrowth for up to 40 days. At low dose levels (2 mg/kg), DP303c showed 47.9% inhibition of tumor growth. By comparison, T-DM1 did not cause tumor regression at all dose levels, and the percent tumor growth inhibition was-28.2%, 16.8%, and 39.9% for 2,4, and 8 mg/kg, respectively.
Example 6: DP303c tumor growth inhibition of mouse xenograft JIMT-1 cancer model compared to T-DM1 and anti-HER 2 biparatopic single chain antibody ADC
The in vivo antitumor activity of DP303c was studied in a mouse xenograft model of human breast cancer. JIMT-1 is a HER 2-positive breast cancer cell line. The JIMT-1 xenograft model is known for its insensitivity to current anti-HER 2 therapeutics, such as trastuzumab and T-DM1 (Li et al, 2016). After tumor cell implantation, tumor-bearing mice were randomly divided into 10 treatment groups (5 mice/group) by single intravenous injection of either blank, DP303c, T-DM1, or biparatopic ADC.
As shown in fig. 6, administration of a single dose of DP303c induced complete tumor regression at all dose levels in the JIMT-1 xenograft model. Tumor regrowth was not found in some of these animals, and they remained tumor-free for up to 4 weeks until the end of the study. In contrast, T-DM1 did not significantly inhibit tumor growth, with TGI% of 4.9%, 16.9% and 16.8% for 8, 116 and 32 mg/kg, respectively. In this study, no significant weight loss or death was observed in all treatment groups.
Example 7: pharmacokinetic and toxicity studies of DP303c
This example describes pharmacokinetic studies of DP303c following single dose injection in rats and after single dose injection in cynomolgus monkeys. This example also describes non-clinical safety studies of DP303c, including non-GLP single dose toxicology studies in rats, non-GLP 29 day dose range finding studies in cynomolgus monkeys, and GLP repeat dose toxicology studies in cynomolgus monkeys. Furthermore, this example describes the in vitro assessment of the stability of DP303c in human and cynomolgus monkey plasma and an in vitro GLP study to evaluate the effect of DP303c formulations on hemolysis and erythrocyte aggregation.
Five week pharmacokinetic study of DP303c in rats (non-GLP) following a single intravenous injection of DP303c
The objective of this study was to determine the pharmacokinetic profiles of DP303c, total anti-HER 2 antibody, free (unconjugated) MMAE and LND1002 (linker MMAE) after a single intravenous administration of DP303c in Sprague Dawley rats at dose levels of 3 mg/kg, 10 mg/kg and 30mg/kg (3/sex/group).
The concentrations of DP303c and total antibody DP001 in rat sera collected from this study were analyzed using an ELISA assay, with a lower limit of quantitation for both DP303c and total antibody DP001 of 0.3125. The concentrations of free MMAE and linker-MMAE (LND002, free payload) were analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method, with a quantitative lower limit of 5.0 pg/mL MMAE and a quantitative lower limit of 0.170 ng/mL LND 1002.
Table 3 below shows the mean pharmacokinetics of DP303c and total antibodiesAnd (4) mechanical parameters. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration; AUC 0-∞ Is the area under the concentration-time curve from time zero to infinity; t is t 1/2 Is the half-life; CL is systemic clearance; v ss Is the steady state distribution volume.
TABLE 3
Figure DEST_PATH_IMAGE016
DP303C C in the dose range of 3 mg/kg to 30mg/kg after a single intravenous bolus DP303C max And AUC ∞ both increased in a dose-proportional manner (fig. 7). The peak concentrations (Cmax) were 73.5, 238 and 630 μ g/mL for the 3, 10 and 30mg/kg dose levels, respectively (Table 3). At 3 mg/kg, the AUC ∞ value is 470 μ g. day/mL, at 10 mg/kg, the AUC ∞ value is 1331 μ g. day/mL, and at 30mg/kg, the AUC ∞ value is 4498 μ g. day/mL. DP303C Clearance (CL) was 6.44, 7.68, and 6.90 mL/day/kg for 3 mg/kg, 10 mg/kg, and 30mg/kg, respectively, and the terminal half-life (t) 1/2 ) For 9.25, 9.79 and 9.42 days, respectively. At 3 mg/kg, steady state distribution volume (V) ss ) 82.9 mL/kg at 10 mg/kg, steady state distribution volume (V) ss ) 104.0 mL/kg at 30mg/kg, steady state distribution volume (V) ss ) It was 90.2 mL/kg. The results show a linear PK of DP303c in rats in the dose range of 3 to 30 mg/kg.
Total antibody C in the dose range of 3 to 30mg/kg after a single intravenous bolus DP303C max And AUC The dose was also increased proportionally (figure 8). At 3, 10 and 30mg/kg, peak concentration of total antibody (C) max ) 71.2, 256 and 701 mug/mL, respectively (Table 3). AUC at 3, 10 and 30mg/kg The values were 515, 1432 and 4428 μ g. day/mL, respectively. Terminal half-life (t) of total antibody at dose levels of 3, 20 and 30mg/kg 1/2 ) Respectively, 9.67, 9.75, and 8.67 days, and Clearance (CL) respectively, 5.86, 7.07, and 6.90 mL/day/kg. Steady State volume of distribution (V) at 3 mg/kg of Total antibody ss ) At a steady state distribution volume (V) of total antibody of 79.1 mL/kg at 10 mg/kg ss ) 96.6mL/kg and a steady state distribution volume (V) of total antibody at 30mg/kg ss ) The concentration was 83.1 mL/kg. The PK profile of total antibody was very similar to that of DP303c, indicating limited deconjugation of MMAE from DP303c in rats.
Serum concentration of MMAE after intravenous administration of DP303C (C) max ) Peak levels were reached at 6 hours post-dose in the 3 and 10 mg/kg dose groups and at 24 hours post-dose in the 30mg/kg dose group, followed by a slow elimination phase (figure 9). The peak concentration of unconjugated MMAE was about 0.03 ng/mL, 0.08 ng/mL, and 0.3 ng/mL for the 3 mg/kg, 10 mg/kg, and 30mg/kg groups, respectively. The exposure of MMAE was DP303c at dose levels of 3 mg/kg, 10 mg/kg and 30mg/kg (AUC) 0-t On a molar basis) of about (0.011%, 0.014%, 0.016%). Serum concentrations of payload LND1002 were not normally detectable in most samples and PK analyses were not performed.
In summary, the PK profile, such as exposure and elimination half-life, between DP303c and total antibody were very similar after a single intravenous administration of DP303c in rats. Linear PK was indicated over the dose range of 3 to 30mg/kg and no sex difference was observed. Serum exposure of free MMAE after a single dose of intravenous D303c was <0.03% DP303 c. Serum concentrations of LND1002 were not normally detectable.
Pharmacokinetic study of DP303c following a single intravenous administration in cynomolgus monkeys
The objective of this study was to determine the pharmacokinetic profile of DP303c after a single intravenous administration of DP303c in cynomolgus monkeys at dose levels of 1.2, 4.0 and 12mg/kg (3/sex/group). In addition, PK of total antibody and free MMAE were also assessed.
Concentrations of DP303c and total antibody DP001 in cynomolgus monkey sera collected from this study were determined by ELISA assay with a lower limit of quantitation for both DP303c and total antibody DP001 of 0.3125 ng/mL. Concentrations of both free MMAE and linker-MMAE (LND002, free payload) in monkey plasma were analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method, with a lower limit of quantitation for MMAE of 0.03 ng/mL and a lower limit of quantitation for LND1002 of 1.0 ng/mL for this study.
Table 4 below shows the mean pharmacokinetic parameters of free MMAE in cynomolgus monkeys after a single intravenous infusion of DP303 c. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration; AUC 0-∞ Is the area under the concentration-time curve from time zero to infinity; NC indicates an uncalculated value.
TABLE 4
Figure 820825DEST_PATH_IMAGE017
C of DP303C at a dose range of 1.2 mg/kg to 12mg/kg after a single intravenous infusion of DP303C max Increase in dose-proportional manner (fig. 10). The peak concentration at 1.2 mg/kg was 30.1 μ g/mL, the peak concentration at 4 mg/kg was 93.5 μ g/mL, and the peak concentration at 12mg/kg was 297.4 μ g/mL (Table 4). AUC at 1.2, 4.0 and 12mg/kg 1300, 6417 and 24834 h. mug/mL, respectively, and Clearance (CL) 0.96, 0.65 and 0.49 mL/h/kg, respectively. DP303c AUC Is greater than dose rate, whereas DP303c Clearance (CL) decreases with increasing dose level, indicating a non-linear PK of DP303c in the dose range of 1.2 to 12mg/kg in monkeys. End-stage half-lives (t) at 1.2 mg/kg, 4.0 mg/kg and 12mg/kg DP303c 1/2 ) 130.1, 136.8 and 118.6 hours, respectively.
Total antibody PK was similar to DP303c PK after a single intravenous infusion of DP303c in cynomolgus monkeys. In the dose range of 1.2 mg/kg to 12mg/kg, total antibody C max Increased in a dose-proportional manner (fig. 11). At 1.2 mg/kg, the peak concentration of the total antibody was 30.8 μ g/mL, at 4 mg/kg, the peak concentration of the total antibody was 103.7 μ g/mL, at 12mg/kg, the peak concentration of the total antibody was 323.7 μ g/mL (Table 4). From 1.2 to 12mg/kg, mean AUC of total antibody The increase in (c) is greater than the dose ratio. At 1.2 mg/kg, mean AUC The value is 1374 h, mug/mL, at 4 mg/kg, average AUC The value is 7333 h. mu.g/mL, at 12mg/kg, the average AUC The value was 27854 h. Mean total antibody AUC for all dose levels Specific average DP303c AUC Not more than 15% larger, indicating limited deconjugation of MMAE from DP303 c. The Clearance (CL) of total antibody decreased from 0.91 mL/day/kg at 1.2 mg/kg, 0.57 mL/day/kg at 4 mg/kg to 0.44 mL/day/kg at 12 mg/kg. At 1.2, 4 and 12mg/kg, the half-life of the total antibody (t) 1/2 ) 123.0, 138.4 and 124.0 hours, respectively.
After administration with DP303c, free MMAE in monkey plasma was not detected or was below the lower limit of quantitation at most time points in the 1.2 mg/kg group. Thus, the pharmacokinetic parameters of free MMAE were not estimated for animals in the 1.2 mg/kg group (table 4). The exposure of free MMAE was very low at both 4 mg/kg and 12 mg/kg. The median time to peak levels of free MMAE in plasma was observed to be 48 hours (fig. 12). C of free MMAE in plasma at 4 and 12mg/kg max 0.059 and 0.135 ng/mL, respectively, and AUC0-t 7.56 and 32.0 h.ng/mL, respectively. In the corresponding dose groups of 4 mg/kg and 12mg/kg, the exposure of free MMAE was only 0.01% of the exposure of DP303 c.
In summary, the mean AUC for DP303c and total antibody after a single intravenous administration of DP303c in cynomolgus monkeys Increasing over the dose scale indicates a non-linear PK of DP303c in cynomolgus monkeys over the dose range of 1.2 to 12 mg/kg. In monkeys, DP303c exposure was lower than total antibody<15%, indicating limited deconjugation of MMAE from DP303 c. The exposure of free MMAE was very low and did not exceed 0.03% of the exposure of DP303 c. No sex difference was observed for DP303c PK in this study.
Preliminary toxicity study (non-GLP) of DP303c after a single intravenous injection in rats
The objective of this study was to assess potential toxicity and determine the Maximum Tolerated Dose (MTD) of DP303c by a single intravenous administration of DP303c in rats followed by a 21 day follow-up period.
Female Sprague Dawley rats (5 per group) were administered either vehicle (control) or DP303c at dose levels of 10, 20, 40, 60 and 100 mg/kg, respectively. Parameters and endpoints evaluated in this study included clinical signs and observations, body weight, food consumption and clinical pathology parameters (hematology) during the 21 day post-dose period. In this study, none of the dose groups were dead or nearly dead. Clinical observations were limited to reduced activity in animals in the highest dose group of 100 mg/kg. Reduced activity started 5 days after dosing, became most pronounced around day 8, and then gradually returned to normal. Mild weight loss was observed in the 100 mg/kg group (about 4% on day 4), and a lower trend in weight gain was found in the other dose groups.
Hematological analysis showed a modest decrease in Red Blood Cells (RBC), hematocrit, and hemoglobin content in animals treated with 60 and 100 mg/kg DP303 c. The total leukocyte and macrophage/monocyte counts were also reduced after DP303c treatment in both high dose groups. Platelet counts decreased slightly, but were not statistically significant. At both high dose levels, all observed adverse effects on hematological parameters were returned to normal 21 days after DP303c treatment. There were no significant treatment-related hematological changes in animals treated with 10, 20, or 40mg/kg DP303 c.
An additional single dose study was conducted to evaluate toxicity at additional dose levels. Sprague Dawley rats (10 per group) were administered either vehicle (control) or DP303c at dose levels of 50, 100 and 200 mg/kg, respectively. Parameters and endpoints evaluated in this study included clinical signs and observations, body weight, food consumption and clinical pathology parameters (hematology) during a post-dose period of 22 days. 200 mg/kg DP303c caused death in the rats. Edema/eschar/ulceration on the chin or neck, temporary abnormal ocular secretion, reduced weight gain and food consumption were observed in the group with a dose level of 100 mg/kg DP303c or higher. An increase in monocytes and a decrease in lymphocytes and eosinophils was observed in the group with a dose level of 50 mg/kg DP303c or higher.
In summary, the main toxicities of a single intravenous administration of DP303c in rats were anemia and leukopenia at high doses (60 and 100 mg/kg), which was reversible after 21 days. The maximum tolerated dose of DP303c in rats was not reached in this study and was not lower than 100 mg/kg after a single intravenous administration.
Discovery of a 29 day dose Range and toxicology Studies (non-GLP) of DP303c after two intravenous injections in cynomolgus monkeys
The objective of this study was to evaluate the potential toxicity and pharmacokinetics (TK) of DP303c in cynomolgus monkeys after intravenous administration of DP303c (2 doses) once every 3 weeks. This study served as a dose range finding to help select doses for GLP studies in subsequent monkeys. Male and female cynomolgus monkeys were administered vehicle (control, 1/sex/group) or DP303c (2/sex/group) at dose levels of 6 (group 2), 20 (group 3), 60 (group 4) and 100 mg/kg (group 5) intravenously by intravenous infusion (for 2 doses) every 3 weeks. The final necropsy was scheduled on day 29, 7 days after the second dose. Due to death and imminent death, only one dose of DP303c was administered in the two high dose groups (60 and 100 mg/kg, groups 4 and 5) with protocol modification.
Toxicology endpoints evaluated in this study included veterinary physical observations, clinical signs, injection site observations, body weight, food consumption, clinical pathology (hematology, coagulation, clinical chemistry, urinalysis), pharmacokinetics, gross autopsy findings, organ weights, and histopathological examination.
DP303 c-related deaths occurred in group 4 (60 mg/kg) and group 5 (100 mg/kg). Two animals at a dose level of 60 mg/kg died on days 10 and 12, respectively, after the first dose. In the 100 mg/kg group, 1 animal died on day 8 and 3 animals were euthanized on day 8 and 10, respectively, due to poor physical condition. Beginning on day 7, these early dead animals exhibited clinical symptoms of reduced activity, self-mutilation, hunched posture, cold skin, prone position, loss of appetite, muscle weakness, watery stool, decreased startle reflex, heavy breathing, and skin necrosis. Weight loss (up to 18%) was found in all of these unplanned dead animals. Hematologic changes included severe reductions in White Blood Cells (WBCs), neutrophils, lymphocytes, monocytes, eosinophils, and reticulocytes at D8 in the 60 and 100 mg/kg groups. At day 15 and later time points, an increase in platelets was found in 2 surviving animals at 60 mg/kg. Gross observations included moderate black discoloration of the lungs in 60 mg/kg, one unplanned necropsy animal, and vaginal hyperplasia in 100 mg/kg, one animal.
No death or imminent death was found in animals treated with low dose levels of DP303c at 6 mg/kg and 20 mg/kg. No DP303 c-related clinical signs were observed in both groups during treatment. On day 15, mild decline in White Blood Cells (WBC), neutrophils and lymphocytes was observed in the 20mg/kg animals, but not at 6 mg/kg. These adverse changes are reversed at later time points. In these 2 treatment groups, there were no DP303 c-related effects on body weight, organ weight, food consumption, blood coagulation and clinical chemistry parameters.
In conclusion, a single dose of DP303c caused mortality or morbidity in all 6 animals at a dose level of 100 mg/kg and in half the animals at a dose level of 60 mg/kg. Animals in these 2 high dose groups had leukopenia characterized by a severe reduction in White Blood Cells (WBCs), neutrophils, lymphocytes, monocytes, and eosinophils. At 60 mg/kg, a moderate dark discoloration of the lungs was found in one of the animals that died off schedule. The reason for unplanned animal death was not very clear, since no histopathological analysis was performed in this study. At low dose levels of 6 mg/kg and 20mg/kg, which continued for 2 doses, all animals survived until the end of the study and no significant clinical signs and clinical pathological changes associated with DP303c were found. Based on these results, DP303c was considered well tolerated in cynomolgus monkeys by intravenous administration of two doses ≦ 20 mg/kg.
The TK profile of DP303c was evaluated by determining the plasma concentrations of DP303c, total antibody and free MMAE. Concentrations of DP303c and total antibody DP001 in cynomolgus monkey sera collected from this study were determined by ELISA assay, with the lower limit of quantitation for DP303c and total antibody DP001 both being 0.3125 ng/mL. Monkey plasma was analyzed for both free MMAE and linker-MMAE (LND002, free payload) concentrations by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method, with a lower limit of quantitation for MMAE of 0.03 ng/mL and a lower limit of quantitation for LND1002 of 1.0 ng/mL for this study.
Table 5 below shows the pharmacokinetic parameters of DP303c in cynomolgus monkeys after intravenous infusion of the first and second doses of DP303 c. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration; AUC 0-∞ Is the area under the concentration-time curve from time zero to infinity; t is t 1/2 Is the half-life; CL is systemic clearance; v ss Is the steady state distribution volume.
TABLE 5
Figure DEST_PATH_IMAGE018
Table 6 below shows the mean pharmacokinetic parameters of free MMAE in cynomolgus monkeys after the first and second dose of intravenous infusion of DP303 c. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration.
TABLE 6
Figure 667558DEST_PATH_IMAGE019
Analysis of pharmacokinetic parameters was performed in groups 2 and 3 in this study because PK sample collections were incomplete at the indicated time points in groups 4 and 5. AUC of DP303c after intravenous infusion of DP303c on day 1 From 12076 h. mug/mL at 6 mg/kg increased above the dose proportion to 78466 h. mug/mL at 20 mg/kg. half-Life (t) of DP303c at 6 and 20mg/kg, respectively 1/2 ) From 86.4 hours to 152 hours, while the Clearance (CL) decreased from 0.525 mL/h/kg to 0.275 mL/h/kg. These data indicate that DP303c is 6 to 20mg/kg in monkeysNonlinear PK over dose range (table 5). For total antibody, a very similar non-linear PK profile was observed (table 5). Total antibody and DP303c Exposure (AUC) ) Are comparable. AUC of total antibody after intravenous infusion of DC303c 0-∞ AUC over DP303c 0-∞ Height of<10 percent. C of MMAE at dose levels of 6 and 20mg/kg max 0.078 and 0.173 ng/mL, respectively (Table 6). Plasma MMAE concentrations reached peak levels between 48 and 96 hours post intravenous infusion (fig. 13). MMAE AUC 0-t 9.29 h.ng/mL at 6 mg/kg and 51.32 h.ng/mL at 20 mg/kg.
DP303c and AUC of Total antibodies from dose levels of 6 to 20mg/kg following intravenous infusion of DP303c on day 22 All increased over the dose ratio (table 5). C of DP303C max And AUC 0-∞ Values were similar between after the first dose and after the second dose. At 6 and 20mg/kg, peak concentration of MMAE (C) max ) 0.045 and 0.210 ng/mL, respectively.
In summary, DP303c, total antibody and PK of free MMAE were analyzed in the 6 and 30mg/kg dose groups. DP303c and AUC of Total antibodies following intravenous infusion of DP303c in cynomolgus monkeys Increase greater than dose ratio, indicating non-linear kinetics. The terminal half-life of DP303c was about 95.3 to 115 hours. MMAE exposure in monkeys was very low compared to DP303 c. Equivalent plasma exposure of intact DP303c and total antibody, along with low levels of free MMAE, indicated negligible unconjugate of MMAE from DP303c in monkeys.
Repeated dose intravenous administration of DP303c every 3 weeks (with a 4-week recovery period) for 5 continuous doses to cynomolgus monkeys Toxicity study (GLP)
The objective of this study was to evaluate the potential toxicity, pharmacokinetics (TK) and immunogenicity of DP303c following intravenous administration of DP303c in cynomolgus monkeys for a total of 5 doses once every 3 weeks. The study also assessed the reversibility of any adverse reactions and possible delayed toxicity during the 4-week treatment-free period. This GLP study was intended to provide key non-clinical safety data to support the first in vivo human (FTIH) phase 1 clinical study of DP303 c.
A total of 20 male and 20 female naive cynomolgus monkeys were selected and enrolled in the study. These animals were randomly assigned to 4 groups based on body weight and sex. Group 1 was a vehicle control (saline, 0 mg/kg), and groups 2, 3 and 4 were DP303c treated, low (6.0 mg/kg), medium (20 mg/kg) and high (40 mg/kg) dose groups, respectively. Each group consisted of five animals of each sex. DP303c and saline were administered via 30 minute intravenous infusion once every three weeks. Three monkeys per sex were assigned to the main study stage per group and the final necropsy was scheduled on day 89, 4 days after the last dose was administered. Two monkeys per sex were assigned to the recovery stage for each group and the recovery necropsy was scheduled on day 113, 4 weeks after the last dose. During the study period, saline and 6 mg/kg DP303c were administered for five doses (on days 1, 22, 43, 64, and 85) as scheduled. DP303c in the intermediate dose group was administered at 20mg/kg in three doses (day 1, day 22, and day 43). For the remaining two doses (day 64 and day 85), the dose was reduced to 12mg/kg due to a single death occurring after the third dose at 20 mg/kg. The dose in this dose group was expressed as 20/12 mg/kg. DP303c in the high dose group was administered at 40mg/kg in two doses (day 1 and day 22). Due to the unplanned animal deaths, the dose of the third dose (day 43) was reduced to 30mg/kg and no more doses were given after the third dose. The dose in this dose group was expressed as 40/30 mg/kg. Necropsy of the surviving animals in the 40/30mg group was scheduled on day 71, 4 weeks after the last dose (recovery period).
Toxicology endpoints evaluated in this study include clinical signs and observations, local tolerance, body weight, food consumption, ophthalmoscopy, body temperature, ECG, clinical pathology parameters (hematology, coagulation, clinical chemistry and urinalysis), lymphocyte phenotype, anti-drug antibody formation and pharmacokinetics (TK), gross autopsy findings, organ weights and histopathological examination.
40/30mg/kg DP303c group were poorly tolerated at the dose level. After the second dose at 40mg/kg, three animals were euthanized at day 20, day 26 and day 29, respectively, due to an imminent death condition. After the third dose of 30mg/kg, one animal died on day 54 and two were euthanized on days 43 and 53, respectively. Symptoms observed include decreased activity, hunched posture, prostate (prostate), cold skin, loss of appetite, salivation, transparent and/or red discharge in the nose, areas of incrustation at various sites of the skin, body stiffness, which begins between day 10 and day 14 in most early-dead animals. Other signs include tearing in the eye, soft and watery stools, rales, slow breathing, dyspnea, and heavy breathing found in some animals. Weight loss was observed at the beginning of the dosing period.
Hematologic changes in the high dose group included a mild to moderate reduction in White Blood Cells (WBCs), lymphocytes, eosinophils, and severe reduction in neutrophils. Other changes include mild to moderate decreases in Red Blood Cells (RBC), hemoglobin, hematocrit, and increases in reticulocytes and platelets. These hematological changes were observed in all animals in the high dose group as early as day 8. An increase in monocyte counts was found at later time points of CP303c treatment. In surviving animals after recovery, the hematological manifestations of anemia and leukopenia return to baseline or abate. Coagulation changes include minimal prolongation of prothrombin time and activated partial thromboplastin time in unplanned dead animals. Clinical chemistry changes included a mild to moderate decrease in albumin and an increase in globulin with a corresponding decrease in albumin to globulin ratio. Other findings include 2-3 fold increase of aspartate aminotransferase and minimal reduction of blood calcium, chloride and creatine. After recovery, the increased aspartate aminotransferase returned to normal.
General observations in the high dose group included a purple or dark discoloration of the lungs, sometimes with lung adhesion to fluid in the chest wall or chest cavity, in 4 out of 6 unplanned dead animals. Increased lung organ weight (associated with bronchi) was also found in these animals. Major microscopic examination in unplanned dead animals found a significant reduction in cell structure including in the thymus, spleen and lymph nodes, and lung inflammation with fibrosis. The cause of unplanned death was attributed to DP303 c-associated lung inflammation and fibrosis, and to depletion of lymphocytes in the thymus, spleen, and lymph nodes. Other important findings include thrombosis in small blood vessels and glomeruli and mild tubular degeneration with or without cast in the kidney, little to mild degeneration/necrosis in the liver, and some single cell necrosis in the skin. After recovery, those histopathological changes in lymphoid organs, lungs and liver were still observed in some surviving animals, but with lower incidence and severity.
At the intermediate dose level of 20/12 mg/kg, one animal died on day 60 after the third dose at 20 mg/kg. All other animals survived until necropsy scheduled at day 89 or day 113. The cause of unplanned death is due to lymphocyte depletion in the spleen and lymph nodes, as well as pulmonary inflammation and fibrosis. Clinical signs in the 20/12 mg/kg group included reduced activity in a minority of animals, loss of appetite, cold skin, transparent or red nasal discharge, soft stools, coughing, and shortness of breath. Hematology findings included minimal reduction in hemoglobin. Clinical chemistry changes included a decrease in albumin to globulin ratio due to a mild increase in globulin. Gross observations included lung discoloration in 4 of 10 animals at terminal necropsy along with lung organ weight gain. The primary microscopic findings included a mild to moderate reduction of lymphocytes in the spleen and/or lymph nodes in 5 of 6 end necropsy animals, and mild to moderate inflammation and fibrosis in the lungs in 4 of 6 animals. At the time of recovery at necropsy, a mild depletion of lymphocytes in lymphoid organs was observed in only 1 of 3 animals, indicating that depletion of lymphocytes in spleen, thymus and lymph nodes was partially reversed. After recovery, pulmonary inflammation and fibrosis were still found in the animals.
All animals treated with DP303c at a low dose level of 6 mg/kg survived well until necropsy scheduled on days 89 and 113. No significant DP303 c-related clinical signs were observed during the primary and recovery periods. There was no DP303 c-related effect on body weight, food consumption, eye examination, body temperature, injection site observations, ECG, hematological parameters, coagulation, and clinical chemistry parameters. In addition, no DP303 c-related organ weight changes and gross findings were found at terminal or recovery necropsies.
In summary, intravenous administration of DP303c at 20mg/kg for 3 doses resulted in 1 death, and after changing the dose from 20mg/kg to 12mg/kg, the other animals survived to scheduled necropsy. Intravenous administration of DP303c at 40/30 mg/mg for 3 doses resulted in mortality or morbidity in 6 of 10 animals. The cause of unplanned death was attributed to DP303 c-related lung inflammation and fibrosis, and to lymphocyte depletion in the thymus, spleen, and lymph nodes. Pulmonary inflammation with fibrosis and mild to moderate depletion of lymphocytes in the spleen and/or lymph nodes were also observed in most of the terminal necropsy animals, but the incidence and severity of these changes were low after recovery. The highest non-severe toxicity dose (HNSTD) recommended under the conditions of this study was 12 mg/kg. Administration of DP303c at 6 mg/mL for 5 doses did not induce any abnormal changes in DP303 c-related clinical and clinical pathological parameters. Thus, the level of No Observed Adverse Effects (NOAEL) of DP303C was considered to be 6 mg/kg/dose (mean C between 4 th dosing intervals) max 137.2µg/mL,AUC 0-t 14149h•µg/mL)。
The TK profile of DP303c was assessed by determining serum or plasma levels of DP303c (whole molecule), total antibody (conjugated and unconjugated antibodies), and MMAE over time. Concentrations of DP303c and total antibody DP001 in monkey sera were determined using an ELISA assay, with a lower limit of quantitation for both DP303c and total antibody DP001 of 0.3125 ng/mL. The concentration of MMAE in monkey plasma was analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method, wherein the lower limit of quantitation of MMAE was 0.03 ng/mL.
Table 7 below shows the pharmacokinetic parameters of DP303c in cynomolgus monkeys after intravenous infusion of DP303 c. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; m indicates male; f indicates female; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration; AUC 0-∞ Is from time zero to infinity under the concentration time curveAccumulating; t is t 1/2 Is the half-life; CL is systemic clearance; v ss Is the steady state distribution volume.
TABLE 7
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Table 8 below shows the pharmacokinetic parameters of total antibodies in cynomolgus monkeys after intravenous infusion of DP303 c. Values are expressed as mean ± standard deviation; [ n ]]Indicating an animal sample number; m indicates male; f indicates female; c max Is the maximum observed concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration; AUC 0-∞ Is the area under the concentration-time curve from time zero to infinity; t is t 1/2 Is the half-life; CL is systemic clearance; v ss Is the steady state distribution volume.
TABLE 8
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Table 9 below shows the pharmacokinetic parameters of free MMAE in cynomolgus monkeys after intravenous infusion of DP303 c. Values are expressed as mean ± standard deviation; [ n ] of]Indicating an animal sample number; m indicates male; f indicates female; t is max Is the time to exist at maximum concentration; AUC 0-t Is the area under the concentration time curve up to the last measurable concentration.
TABLE 9
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DP303C C after intravenous infusion of DP303C on day 1 max The dose was proportionally increased from 6 mg/kg of 131.7 (male)/147.4 (female) μ g/mL to 40 mg/mL of 935.4(M)/943.6(F) μ g/mL (FIGS. 14A and 14B). Mean AUC 0-t Increasing from 12668 (M)/13463 (F) μ g.h/mL at 6 mg/kg to 126795 (M)/132131 (F) μ g.h/mL at 40mg/kg in excess of the dose ratio (Table 7). Clearance of DP303c for 6-, 20-, and 40-mg/kg dose levelsThe rates (CL) were 0.46 (M)/0.44 (F), 0.33 (M)/0.34 (F) and 0.28 (M)/0.26 (F) mL/day/kg, respectively, and the half-lives (t floor) were 115.1 (M)/103.0 (F), 168.3 (M)/159.8 (F) and 158.3 (M)/162.3 (F) hours, respectively. Reduction of CL and t with increasing DP303c dose 1/2 The increase in (c) is parallel. These data indicate a non-linear PK of DP303c and indicate the presence of troughs that became saturated between single intravenous doses of 6 and 40 mg/kg.
DP303C C in the 6 mg/kg dose level group after intravenous administration of DP303C on day 64 max And AUC 0-t Are 134.3 (M)/140.1 (F) μ g/mL and 14031 (M)/14266 (F) μ g.h/mL (FIGS. 14A and 14B). DP303C C after the first dose and after the 4 th dose on day 64 max And AUC 0-t Are similar, indicating that there was no accumulation of DP303c exposure after its Q3W administration. t is t 1/2 And CL values were approximately comparable between the first dosing interval and the 4 th dosing interval. In addition, similar DP303c concentration-time curves were observed in males and females, indicating similar systemic exposure in both sexes (table 7).
Total antibody PK was approximately similar to DP303c PK. Mean total antibody C after a single intravenous infusion of DP303C on day 1 max Dose was proportionally increased from 144.5 (M)/157.2 (F) μ g/mL at 6 mg/kg to 935.4 (M)/833.7 (F) μ g/mL at 40 mg/mL (FIGS. 15A and 15B). Mean AUC of Total antibody 0-t The value increased from 15039 (M)/15338 (F) μ g.h/mL at 6 mg/kg slightly above the dose ratio to 126795/113875 μ g.h/mL at 40mg/kg (Table 8). CL for total antibody was 0.38 (M)/0.38 (F), 0.29 (M)/0.27 (F), 0.28 (M)/0.30 (F) mL/day/kg, and t for 6-, 12-and 30-mg/kg dose levels, respectively 1/2 123.7 (M)/108.8 (F), 179.0 (M)/178.1 (F) and 158.3 (M)/177.6 (F) hours, respectively. C of Total antibody after first dose max And AUC 0-t The mean values of (A) were comparable to the mean values after the 4 th dose at 6 mg/kg, indicating that there was no accumulation of total antibody exposure after multiple doses.
Peak concentrations of free MMAE in plasma were observed between 72-317 hours at all dose levels after intravenous infusion administration of DP303c on day 1 (fig. 16A and 16B). Average C of free MMAE max From 0.0635 (M)/0.0641 (F) ng/kg at 6 mg/kg to 0.3838 (M)/0.4878 (F) ng/kg at 40mg/kg (Table 9). AUC of free MMAE 0-t Also as the dose increased from 13.08 (M)/8.94 (F) to 135.12 (M)/141.12 ng.h/mL. Peak concentrations of free MMAE in plasma were found between 24-96 hours at all dose levels after intravenous infusion of DP303c on day 43 or day 64 (fig. 16A and 16B). C of free MMAE at 6-, 12-and 30-mg/kg dose levels of DP303C max The values were 0.0529 (M)/0.0616 (F), 0.3845 (M)/0.1727 (F) and 0.6787 (M)/0.8637 (F) ng/kg, respectively (Table 9). Average AUC at 6 mg/kg 0-t 7.11 (M)/14.15 (F) ng.h/mL, mean AUC at 12mg/kg 0-t 114.52 (M)/53.53 (F) and an average AUC at 30mg/kg 0-t 151.37 (M)/143.39 (F) ng.h/mL.
An anti-drug antibody (ADA) test was performed in this study to evaluate ADA against DP303 c. In GLP repeat dose toxicology studies, bridging immunoassays are used to screen serum samples from cynomolgus monkeys for anti-drug antibodies (ADA) against DP303 c. In this assay, biotin-labeled DP303c, ADA, and DP303c formed a bridge complex that was quantified using streptavidin-HRP. The signal to background ratio (S/B) for each sample was compared to the cut-off factor. Any sample with an S/B equal to or above the cut-off factor was considered positive. The sensitivity of the assay was determined to be 14.27 ng/mL. All samples were found to be negative and ADA was not detected.
In summary, plasma exposure of DP303c and total antibodies increased more than dose-proportionally after intravenous infusion administration of DP303c in cynomolgus monkeys on days 1 and 43/64, indicating non-linear kinetics. No accumulation of DP303c or total antibody was observed after Q3W was administered DP303 c. The terminal half-life of DP303c was 120.6 to 162.5 hours. Equivalent plasma exposure of intact DP303c and total antibody together with very low levels of free MMAE indicated negligible unconjugate of MMAE from DP303c in monkeys. Male and female cynomolgus monkeys showed similar concentration-time curves for all analytes.
Plasma stability studies of DP303c in human and cynomolgus monkey plasmaExcellent (GLP)
The present study is currently evaluating the in vitro stability of DP303c in pooled plasma from human and cynomolgus monkeys. DP303c was incubated at a concentration of 100 μ g/mL in human or monkey plasma for 96 hours at 37 ℃. Samples were collected at 0,4, 24, 48, 72 and 96 hours post incubation and then analyzed for DP303c, free MMAE and total DP001 (naked DP001 plus DP303 c).
Concentrations of DP303c and total antibody DP001 in cynomolgus monkey and human plasma were analyzed using an ELISA assay with a lower limit of quantitation for DP303c and total antibody DP001 of 0.3125 ng/mL. The concentration of MMAE in monkey plasma was analyzed by a tandem liquid chromatography/mass spectrometry (LC-MS/MS) method, wherein the lower limit of quantitation of MMAE was 0.03 ng/mL.
Table 10 below shows the stability of DP303c in monkey and human plasma at 37 ℃.
Watch 10
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Table 11 below shows the formation of free MMAE in monkey and human plasma during 96 hours at 37 ℃. Values are expressed as mean ± standard deviation; the NC representative value is not calculated; a indicates that the free MMAE concentration in 2 out of 3 samples is below the lower limit of quantitation (LLOQ).
TABLE 11
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During the 96 hour test period, the concentration changes of DP303c and DP001 were very small in monkey (no change) and human (about 10% reduction) plasma (table 10). The ratio between DP303c and DP001 ranged from 0.96 to 1.11 at all time points. Consistent with this result, the free MMAE concentration in monkey and human plasma was very low by the end of the study: 1.318 and 0.752 ng/ml in monkey and human plasma, respectively (Table 11). Free MMAE was about 0.132% and 0.073% of DP303c concentration in monkey and human plasma. All these data indicate that DP303c has very good conjugation stability in plasma.
Investigation of the hemolytic potential of DP303c in the blood of human and Macaca fascicularis (GLP)
The objective of this study was to evaluate the potential effect of DP303c preparations on hemolysis and erythrocyte aggregation in human and cynomolgus monkey blood. Human or cynomolgus monkey erythrocytes were incubated with DP303c at concentrations of 0.2, 0.4, 0.6, 0.8 and 1mg/mL for 3 hours at 37 ℃. No hemolysis and coagulation was observed with DP303c by visual or spectrophotometric analysis during the 3 hour test period, indicating that DP303c did not cause hemolysis and aggregation of human and monkey red blood cells.
Discussion of the related Art
DP303c demonstrated very good in vitro stability in human and monkey plasma. After 96 hours of incubation in human plasma, 90% of DP303c were intact molecules. In the dose range of 3 to 30mg/kg, DP303c exhibited linear PK in rats with a terminal half-life of 9 days. PK parameters between DP303c and total antibody were very similar in all doses of rats, indicating limited deconjugation of MMAE from DP303 c. In all 3 single and repeated dose cynomolgus studies, DP303c exhibited non-linear PK in the dose range of 1.2 to 40mg/kg with a terminal half-life of 4 to 7 days. The comparable exposure of DP303c and total antibody in cynomolgus monkeys also indicates that MMAE was not significantly deconjugated in monkeys. Plasma levels of MMAE were very low in monkeys after intravenous administration of DP303 c. No sex differences were observed in the PK profile of DP303c, total antibody or free MMAE in all animal studies. In repeated dose GLP studies, no accumulation of DP303c or total antibody was observed after Q3W administration of DP303 c.
In both the non-GLP and GLP studies, dose levels ≧ 40/30mg/kg DP303c were intolerable in cynomolgus monkeys, leading to death and imminent death of the animals. Dose-dependent leukopenia and anemia characterized by RBC mass, WBCs, neutrophils, eosinophils, and lymphopenia were also found in both studies. In GLP repeat dose toxicology studies, the major dose-limiting toxicities were dose-dependent lymphocyte depletion and pulmonary toxicity in spleen, thymus and lymph nodes. Clinical symptoms of pulmonary toxicity include cough, dyspnea, and heavy breathing, with a general observation of purple or dark discoloration of the lungs, increased lung organ weight, and pulmonary inflammation with fibrosis. However, lung toxicity was not clearly noted in the 29 day non-GLP study as assessed by gross observation and lung organ weight. A possible explanation might be that it took longer to develop significant lung inflammation with fibrosis and lung injury after DP303c treatment, since in the 29 day non-GLP dose range finding study, the planned extrinsic animal death occurred at day 10 or earlier. Other significant histopathological findings in GLP repeat dose toxicology studies include mild tubular degeneration with or without cast (cast) in the kidney, minimal or mild degeneration/necrosis in the liver, and mild single cell necrosis in the epidermis in the skin or injection site.
In a 29 day non-GLP dose range finding study, a dose level of 20mg/kg DP303c was well tolerated. In the GLP repeat dose toxicology study, all animals survived 20/12 mg/kg DP303c, except for one planned animal death that occurred before the dose was changed from 20 to 12 mg/kg. In a small number of animals, clinical signs at 20/12 mg/mL were limited to red discharge in the nose, loss of appetite, soft stools, cold skin, reduced activity, coughing, and shortness of breath. Histopathological findings were similar to those under 40/30mg/kg DP303c, but were generally milder and partially reversible after recovery. Based on these data, the highest non-severe toxic dose (HNSTD) of DP303c in monkeys was considered to be 12 mg/kg/dose IV once every 3 weeks. In both the 29 day non-GLP dose range finding study and the GLP repeat dose toxicology study, all animals survived at the low dose level of 6 mg/kg DP303c, without DP303 c-related clinical signs and clinical pathology findings. Furthermore, no signs of DP303 c-related cardiotoxicity were observed at any dose, as assessed by ECG. Thus, the level of No Observed Adverse Effects (NOAEL) of DP303c in monkeys was determined to be 6 mg/kg administered intravenously once every three weeks.
In addition to cynomolgus monkeys, the non-clinical safety of DP303c was also evaluated in rats in a non-GLP single dose toxicology study (study DP-15-0818). In this study, there was no death or imminent death in all groups. Major adverse reactions include a reduction in leukocytes, macrophages/monocytes, hematocrit and hemoglobin at high doses (60 and 100 mg/kg) of DP303 c. The hematological changes observed returned to normal levels 21 days after DP303c treatment. Thus, in this study, the maximum tolerated dose of DP303c was not reached in rats and was determined to be not less than 100 mg/kg following a single intravenous administration. The difference in tolerance of DP303c in monkeys and rats was likely due to differences in antigen distribution and binding.
In summary, the major toxicity of DP303c in non-clinical safety studies was anemia, leukopenia, depletion of lymphocytes in the thymus, spleen and lymph nodes, and lung inflammation and fibrosis. These adverse effects are dose-dependent, reversible, and monitorable in animals. Thus, based on the completed non-clinical safety pack, the ICH S9 guidelines, and the FDA publication (Saber and Leighton, 2015), a starting dose of 0.6 mg/kg was proposed for the first human trial with DP303 c. Body weight was used for scaling based on 1/10 at 6 mg/kg in cynomolgus monkeys th NOAEL calculated the initial dose.
Example 8: DP303c PK assay for first in vivo human (FTIH) studies
Based on the assays that are currently validated for non-clinical studies, the following PK assays for first in vivo human (FTIH) studies were developed and validated: 1) an ELISA assay for quantifying the level of DP303c in human serum samples; 2) an ELISA assay for quantifying the level of DP001 (total antibody) in human serum samples; 3) liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to quantify the level of free MMAE warheads in human serum samples. Experiments were performed to evaluate the following assay parameters: 1) upper and lower quantitative limits; 2) accuracy and precision; 3) specificity; 4) selectivity; 5) dilution linearity; and 6) sample stability. Immunoassays and hierarchical immunogenicity testing methods were used for ADA detection to support the FTIH clinical trial. The samples will be screened first and once the test sample is confirmed to be positive for the presence of ADA, the specificity of ADA will be assessed. Will be directed to 1) detecting a limit; 2) accuracy; 3) performing truncation; and 4) drug tolerance assessment ADA assay. The sample matrix will be serum.
The dose escalation regimens for the DP303c FTIH study were 0.6, 1.2, 2.0, 3.0 and 4 mg/kg intravenous infusions once every 3 weeks (Q3W). The optional escalation to higher dose levels achieved by increasing up to 25% from the previous dose level per cohort may be assessed based on safety and clinical activity data from the study. DP303c dose selection was based on the amount of safety margin from non-clinical safety studies and FDA publications (Saber and Leighton, 2015) and ICH S9. Using body weight for scaling, the amount of safety margin was estimated based on 1/10 at 6 mg/kg of no adverse effect observed level (NOAEL) in cynomolgus monkeys. The body surface area was used for scaling, and the margin of safety was also calculated based on 1/6 for the highest non-severe toxic dose (HNSTD) of 12mg/kg in cynomolgus monkeys. A starting dose of 0.6 mg/kg is expected to have a margin of safety of 10 (based on 6 mg/kg of HED NOAEL) and 6.5 (based on 3.87 mg/kg of HED HNSTD) (Table 12). The highest dose of 4 mg/kg is expected to have a margin of safety of 1.5 (based on HED NOAEL) and 1 (based on HED HNSTD).
TABLE 12 predicted margin of safety for proposed clinical doses of DP303c
Figure 30034DEST_PATH_IMAGE025
Example 9: safety, pharmacokinetic, immunogenicity, and antitumor Activity Studies of DP303c
This example describes a phase 1a/1b multicenter, open label, dose escalation/decrementation and dose extension study to evaluate the safety, pharmacokinetics, immunogenicity and antitumor activity of DP303c in subjects with selected advanced solid tumors expressing HER 2. The main objective of this study was to assess the safety and tolerability of DP303c in subjects with HER 2-positive advanced solid tumors that are refractory to standard therapy or in the absence of standard therapy, and to determine the Maximum Tolerated Dose (MTD) and/or the recommended phase 2 dose (RP2D) for DP303 c. A secondary objective of this study was to assess the antitumor activity of DP303c in subjects with HER 2-positive breast or gastric cancer, to determine the Pharmacokinetic (PK) profile of DP303c, and to determine the immunogenicity of DP303 c.
Stage 1 a: dose escalation/decrementation
The phase 1a portion of the study enrolled subjects with HER 2-positive advanced solid tumors. Dose level cohorts of 3 subjects each were treated. The dose levels to be studied in phase 1a (DL) were: DL-1: 0.3 mg/kg; DL 1: 0.5 mg/kg or 0.6 mg/kg; DL 2: 1mg/kg or 1.2 mg/kg; DL 3: 2.0 mg/kg; DL 4: 3.0 mg/kg; DL 5: 4.0 mg/kg. The starting dose (DL 1) was 0.5 mg/kg or 0.6 mg/kg. For the first dose, the first dose is administered by intravenous infusion in physiological saline over 60 (± 10) minutes; subsequent infusions can be administered over 30 (± 5) minutes if tolerated.
The present study used a traditional 3+3 design that was widely used in phase 1 studies to determine MTD and as a basis for selecting RP 2D. The initial dose was 0.6 mg/kg, one tenth of the NOAEL (no adverse effect level observed) in a monkey definitive repeat toxicity study. The 4.0 mg/kg dose is lower than the dose associated with severe toxicity in monkeys (20 mg/kg, which is comparable to the 6.7 mg/kg human dose after isovelocity scaling).
In a dose-escalation design, dose-limiting toxicity (DLT) is evaluated over an initial 21 day and later period to determine dose escalation or dose decrementation. If the 4.0 mg/kg dose level does not exceed the MTD, then the dose escalation may continue to increase up to 25% from the previous dose level until the MTD (. gtoreq.2 DLT) is exceeded at the dose level. DLT was defined by NCI CTCAE v 4.03 as a study drug-related AE, or a study drug-related and clinically significant laboratory abnormality that occurred during the first 21-day cycle of DP303c, and met the following criteria: grade 4 neutropenia (absolute neutrophil count [ ANC)]<500/mm 3 ) Is continued for>7 days; accompanied by fever (temperature)>38.0 ℃) febrile neutropenia (ANC)<1000/mm 3 ) Continue to sustain>1 hour; grade 4 thrombocytopenia (platelet count)<250,000/mm 3 ) Is continued for>2 days; grade 3 or more thrombocytopenia (platelet count) with clinically significant bleeding<50,000/mm 3 ) (ii) a Pneumonia of grade 2 or more(ii) a Any other grade 3 or more non-hematologic toxicities not including sub-optimal treatment of nausea, vomiting and diarrhea, alopecia and/or transients ((R))<1 week) grade 3 fatigue; the optimally treated nausea, vomiting or diarrhea is grade 3 or greater, which lasts for more than 72 hours; LVEF<40% or LVEF drop from baseline by 10% or more and LVEF<45 percent; treatment delay due to DP303 c-related toxicity>And 21 days.
The onset of DLT-like toxicity after cycle 1 (C1) was continuously assessed and incorporated into future decisions regarding further dose escalation/decrementation. An AE is not considered a possible DLT if existing evidence indicates that a relationship to study treatment is logically impossible, medically unreasonable or highly impossible due to explicit surrogate explanations.
Decisions regarding dose escalation are made based on cumulative safety data including adverse event reports, vital signs, laboratory results, ECG, and results of LVEF and pulmonary function tests. During the DLT evaluation period in C1, the following rule was used to determine whether dose escalation was appropriate: initially 3 subjects will be enrolled in each cohort; if none of the first 3 subjects in the cohort experienced DLT, up to 3 new subjects will be enrolled in the cohort at the next dose level; if 1 of the first 3 subjects in the cohort experienced a DLT, the cohort would be expanded to as many as 6 subjects. If no additional subjects in the 6 subjects in the cohort develop DLT, up to 3 new subjects will be enrolled in the cohort for the next dose level; if 2 or more subjects in the 3 or 6 subject cohort experience a DLT, then the dose level is above the MTD and dose escalation will cease, 3 additional subjects will be enrolled and evaluated for DLT at the previous dose level unless 6 subjects have already been evaluated at that dose level; when a dose above the MTD has been tested, the highest dose that experiences DLT in less than 2 of 6 subjects (i.e. <33%) will be considered the MTD.
Dose escalation decisions for each cohort were made after the last subject had completed the DLT observation period or experienced DLT and had reviewed all relevant safety data. Up to 3 subjects could be added to the cohort to more thoroughly assess potential safety signs even though no DLT was observed. In some cases, treatment-related AEs that occurred after the 21-day DLT assessment period were considered for dose escalation/decrementation decisions.
Stage 1 b: dose extension
RP2D is a dose expected to be tolerated for repeated administration. Upon review of clinical safety data, RP2D may be lower than the MTD. After determination of the MTD and selection of RP2D, the phase 1b dose expansion portion of the study will begin. During the extension, approximately 10 subjects each were enrolled into 2 defined cancer types: breast and gastric cancers (including adenocarcinoma at the gastroesophageal junction). For both tumor types, HER 2-positive was defined as: HER2 IHC 2+ and ISH positive or IHC 3 +.
After 10 subjects (summed over the two dose-extended cancer types) have received the first study treatment cycle (3 weeks/21 days), SMC will review all available safety data and assess whether any modification to the dosing regimen or study design is necessary.
For each extended cancer type, if at any time ≧ 3 subjects experience a DLT-like AE or other unacceptable toxicity, further enrollment will be suspended, awaiting SMC review. SMC may suggest enrolling subsequent subjects at the next lower or intermediate dose level.
Inclusion and exclusion criteria
Approximately 54 subjects from different sites were enrolled, up to 15-30 during dose escalation/decline (phase 1 a) and up to 20-24 during dose escalation (phase 1 b), with approximately 10 subjects in each cancer type. Additional subjects may be enrolled to address withdrawal and ensure that a minimum number of DLT evaluable subjects during dose escalation. Subjects were eligible for inclusion in the study only if all of the following criteria were applicable:
1. signing an informed consent form before researching the relevant program;
2. male or female 18-75 years old;
3. diagnosis of an advanced, HER 2-positive Estrogen Receptor (ER) malignancy that has progressed or does not exist following standard therapy. Subjects who have been previously treated with HER2 targeted therapy (such as trastuzumab, pertuzumab, lapatinib, or enrmetuzumab) are eligible. HER 2-positive was defined as IHC 2+ and ISH positive or IHC 3 +: for stage 1a, the subject may have any type of solid tumor, provided that it is IHC 2+ or 3+ positive or ISH-positive for HER 2; for stage 1b, the subject must have breast or gastric cancer that is IHC 2+ or 3+ positive or ISH-positive for HER 2; the HER2 test must be conducted in a laboratory approved by the american academy of pathology (CAP) using an FDA-approved or validated assay.
4. ECOG performance status 0 to 1 and expected survival time greater than 3 months;
5. the subject must have laboratory values within the following limits: ANC ≥ 1.5 x 10 9 L; platelet count is not less than 100 x 10 9 L; the hemoglobin is more than or equal to 9 g/dL; serum creatinine is in a normal range or the creatinine clearance rate is more than or equal to 60 mL/min; serum total bilirubin ≦ 1.5 x ULN (up to 3 x ULN in subjects with Gilbert syndrome); AST (SGOT) and ALT (SGPT). ltoreq.2.5X ULN (or. ltoreq.5X ULN for subjects with liver metastases); PT/INR and APTT are less than or equal to 1.5 x ULN;
6. disease measurability: stage 1 a: measurable or evaluable disease; stage 1 b: the disease must be measurable (according to RECIST 1.1);
7. WOCBP must have a negative pregnancy test prior to entry into the study;
8. WOCBP and male subjects must agree to use adequate contraception at least 12 weeks from study entry to the last dose of study medication;
9. subjects who have recently received anti-tumor systemic therapy require a period of clearance. The time period prior to the first dose DP303c planned by the subject must be at least 28 days or 5 half-lives, whichever is shorter. This applies to both research and approved therapies. Anti-tumor therapies include chemotherapy, immunotherapy, targeted therapy, endocrine therapy, radiotherapy (except for local radiotherapy for pain relief, 14 days after treatment).
Subjects are prohibited from receiving the following therapies during the screening and study treatment periods: anti-cancer systemic chemotherapy, hormonal therapy or immunotherapy; performing selective surgery/dental treatment after discussion with a medical supervisor according to negotiation with an applicant; (ii) a study agent other than DP303 c; radiotherapy (except palliative radiotherapy for disease-related pain and consulting medical supervisors of sponsors); a radioactive/toxin immunoconjugate.
Subjects meeting any of the following criteria will not be eligible for participation in the study:
1. pregnant or lactating women;
2. refusing to use an effective contraceptive method (for details, see inclusion criteria);
3. no recovery from AEs other than alopecia due to previously administered agents or radiation therapy (i.e., ≦ grade 1 or at baseline);
4. a history of <40% cardiac dysfunction while receiving trastuzumab therapy;
5. subjects with a history of allergy to any component of DP303c (trastuzumab analog, MMAE, sodium citrate dihydrate, citrate monohydrate, polysorbate 20, sucrose, etc.);
6. a history of unstable Central Nervous System (CNS) metastases or epileptic disorders associated with malignancy; however, subjects who were directed to previous CNS metastasis treatments and have been asymptomatic for a period of at least 4 weeks while the steroids and anticonvulsants were discontinued may participate in the study;
7. a history of interstitial lung disease, previous or active lung infection, or inflammation (pneumonia);
8. oxygen needs to be supplemented;
9. history of congestive heart failure, unstable angina, unstable atrial fibrillation, or arrhythmia. Subjects with the following types of cardiac injury at enrollment: new york heart association class III or IV heart disease; uncontrolled angina, congestive heart failure or myocardial infarction within 6 months prior to enrollment; LVEF <50% obtained by Echocardiography (ECHO) or multi-gated acquisition (MUGA) scanning; QT interval prolongation (> 450 ms in men, 470 ms in women);
10. the cumulative dose of the anthracycline is more than or equal to 360mg/m in the first 90 days of the study 2 Doxorubicin or an equivalent;
11. peripheral neuropathy was grade 2 or higher (NCI CTCAE v 4.03);
12. unmanageable electrolyte imbalances including hypokalemia, hypocalcemia or hypomagnesemia (based on NCI CTCAE v 4.03, grade 2 or higher);
13. any uncontrolled intercurrent disease, infection or other conditions that may limit study compliance or interfere with evaluation;
14. a subject with evidence of active infection comprising: subjects treated with antibiotics for active infection at enrollment; a subject with evidence of active hepatitis c or chronic active hepatitis b; a subject known to be diagnosed with Human Immunodeficiency Virus (HIV) infection/acquired immunodeficiency syndrome (AIDS);
15. patients treated with CYP3A inhibitors (drugs that increase the AUC of a particular CYP substrate by more than or equal to 5-fold, such as imidazopidil, verapamil, diltiazem, nefazodone, clarithromycin, telithromycin, dactinomycin, erythromycin, fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole tablets, eptivir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, boceprevir, inconvo, telaprevir, conitane, idalaisi) or strong CYP3A inducers (avasimibitartb, phenobarbital, phenytoin, carbamazepine, rifampin, rifabutin, enzalutamide, mitotane, hypericum perforatum) within 14 days of first administration;
16. other serious or poorly controlled diseases or conditions that would interfere with the assessment of a critical study endpoint or that would be considered by the investigator to place the subject at risk of not participating in the study.
Dose modification
Dose reduction and/or delay may be considered for the following cases:
1. in the event of a DLT during cycle 1 or an AE meeting DLT criteria or an AE deemed unacceptable by the investigator at or after cycle 2, subjects were either discontinued from the study or returned to dosing at a reduced dose level after a return from DLT to grade 0, grade 1 or baseline in a subsequent cycle.
2. In the case where a drug-related AE has not returned to grade 0, 1, or baseline at the end of the 21-day cycle, dosing may be resumed at full or reduced doses if the AE returns by day 42 post-dose. Subjects were discontinued from the study if AE had not recovered by day 42.
3. In the event that the medication is missed for regulatory reasons (e.g., site shut-down during holidays, subject vacation, intercurrent illness), the medication can be resumed at the next feasible time. Subjects were discontinued from the study if dosing could not be resumed by day 42 post-dose.
Allowing the dose to be reduced to the dose level prescribed by the next lower regimen. Patients enrolled at the lowest dose level (DL 1: 0.6 mg/kg) can reduce their dose to 0.3 mg/kg. If the subject cannot tolerate a reduced dose level, he/she should be stopped from the study. Dose escalation in subjects was not allowed.
Evaluation of the study
Efficacy was assessed by tumor response after cycle 2 was completed and at the end of every other cycle until the study was stopped. Safety was assessed by measurements of physical examination, vital signs, Electrocardiogram (ECG), Left Ventricular Ejection Fraction (LVEF), pulmonary function test and high resolution CT of the chest.
The measured values of the main targets are: adverse Events (AE), Severe Adverse Events (SAE), dose-limiting toxicity (DLT), changes in laboratory parameters from baseline, Vital Signs (VS) and Electrocardiogram (ECG). All AEs and SAEs will be collected from ICF signup until 30 (± 5) days after the last dose of DP303 c. Events meeting the AE definition are:
1. any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) or other safety assessments (e.g., ECG, radiology scans, vital sign measurements), including those worsening from baseline, considered clinically significant (i.e., not associated with progression of the underlying disease) in the researcher's medical and scientific judgment.
2. Exacerbations of chronic or intermittent pre-existing disease, including increased frequency and/or intensity of the condition.
3. A new condition detected or diagnosed after treatment administration is investigated, even though it may already exist before the study began.
4. Signs, symptoms or clinical sequelae of suspected drug-drug interactions.
5. The signs, symptoms or clinical sequelae of treatment or suspected overdose with the drug were investigated. Overdose by itself will not be reported as AE/SAE unless it is a deliberate overdose with potentially suicidal/self-disabling intent. Such overdosing should be reported regardless of the sequelae.
6. "lack of efficacy" or "failure to expect a pharmacological effect" is not reported as an AE or SAE per se. Such a situation would be included in the efficacy assessment. However, if the signs, symptoms and/or clinical sequelae resulting from the lack of efficacy meet the definition of AE or SAE, they will be reported as AE or SAE.
Events that do not meet the AE definition are:
1. any clinically significant abnormal laboratory findings or other abnormal safety assessments associated with the underlying disease unless judged by the investigator to be more severe than expected in the subject's condition.
2. The disease/disorder in question or the expected progression, sign or symptom of the disease/disorder in question, unless the subject's condition is more severe than expected.
3. Medical or surgical procedures (e.g., endoscopy, appendectomy): the condition leading to the surgery is AE.
4. A situation where no adverse medical event has occurred (social and/or convenient admission).
5. Expected diurnal fluctuations of pre-existing diseases or conditions, which did not worsen, were present or detected at the beginning of the study.
The measured values of the secondary targets are: optimal overall response and disease control rate based on RECIST 1.1; duration of reaction and progression-free survival (PFS); individual subject DP303c serum concentrations and other analytes, as well as derived PK parameters, at specified time points following administration of DP303 c; number (%) of subjects who produced detectable anti-drug antibody (ADA). Pharmacokinetics will be assessed by measuring DP303c concentration, total antibodies in serum, and MMAE derivatives and free MMAE concentration in plasma. Blood samples will be collected from all subjects for measurement of DP303c concentration in serum or plasma on days 1, 2,4, 8, 15 of cycle 1 and cycle 2 and day 1 of all subsequent cycles. Anti-drug antibodies against DP303c in serum will be measured in blood samples collected on day 1 of each treatment cycle. Antitumor activity will be analyzed by Objective Response Rate (ORR), Disease Control Rate (DCR), duration of response, and Progression Free Survival (PFS). ORR is defined as the ratio using RECIST 1.1 criteria ([ confirmed Complete Response (CR) + confirmed Partial Response (PR) ]/N evaluable). Ratios that require no confirmation to achieve CR or PR will also be provided. ORR will be summarized using descriptive statistics. Disease Control Rate (DCR) [ CR + PR + disease Stability (SD)/N evaluable ] will be similarly summarized. The reaction duration and PFS will be calculated using the Kaplan-Meier method.
Initial result of 1a
To date, 10 subjects were enrolled in the phase 1a study to assess the safety and efficacy of DP303c treatment in the subjects. All enrolled subjects had HER 2-positive cancer and had received HERCEPTIN prior to the study ® And (6) treating. To date, five subjects have been withdrawn and are still participating in the study. For the dose escalation study design, one subject was administered 0.5 mg/kg DP303c, and three subjects were each administered 1, 2, or 3 mg/kg DP303 c. The dose was administered by intravenous infusion once every three weeks.
DP303 c-related toxicity was evaluated. At a dose of 2mg/kg, one subject showed grade 2 ocular toxicity with blurred vision. At a dose of 3 mg/kg, one subject showed grade 3 ocular toxicity with blurred vision. After both patients suspended dosing (i.e., they both skipped a scheduled dose), all observed toxicities resolved and treatment with DP303c resumed. After the pause, the patient who had previously received 3 mg/kg drops to 2 mg/kg.
Efficacy was evaluated using RECIST 1.1 criteria (see Eisenhauer, e.a. et al)Eur J Cancer2009 Jan;45(2): 228-47). At doses of 1, 2 and 3 mg/kg, one patient from each dose level showed a partial response (30% or greater reduction in tumor size). A summary of toxicity and efficacy results is provided in table 13 below, where "PD" indicates disease progression, "PR" indicates partial response, and "SD" indicates disease stability.
Table 13: summary of efficacy and toxicity results at stage 1a
Figure DEST_PATH_IMAGE026
Figure IDA0003624257010000011
Figure IDA0003624257010000021
Figure IDA0003624257010000031
Figure IDA0003624257010000041
Figure IDA0003624257010000051
Figure IDA0003624257010000061
Figure IDA0003624257010000071
Figure IDA0003624257010000081

Claims (21)

1. A method of treating cancer in an individual comprising administering to the individual an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an anti-HER 2 antibody and a conjugate moiety comprising a toxin, wherein the anti-HER 2 antibody comprises a glycosylated Fc region comprising an endogenous receptor glutamine residue, wherein the conjugate moiety is conjugated to the receptor glutamine residue, and wherein the antibody-drug conjugate is administered at a dose of no more than about 8 mg/kg.
2. The method of claim 1, wherein the cancer is a solid cancer.
3. The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urinary tract cancer, and lung cancer.
4. The method of any one of claims 1-3, wherein the antibody-drug conjugate is administered at a dose of no more than about 6 mg/kg.
5. The method of any one of claims 1-3, wherein the antibody-drug conjugate is administered at a dose of about 1mg/kg to about 2mg/kg or about 2mg/kg to about 3 mg/kg.
6. The method of claim 5, wherein the antibody-drug conjugate is administered at a dose of about 1.0 mg/kg, about 2.0 mg/kg, or about 3.0 mg/kg.
7. The method of any one of claims 1-6, wherein the antibody-drug conjugate is administered intravenously.
8. The method of any one of claims 1-7, wherein the antibody-drug conjugate is administered about once every three weeks, about every other week, or about once a week.
9. The method of any one of claims 1-8, wherein the individual is a human.
10. The method of any one of claims 1-9, wherein the Fc region of said anti-HER 2 antibody is N-glycosylated.
11. The method of any one of claims 1-10, wherein said receptor glutamine residue is Q295 in the heavy chain of an anti-HER 2 antibody according to EU numbering.
12. The method of any one of claims 1-11, wherein each heavy chain of said anti-HER 2 antibody is conjugated to said conjugate moiety.
13. The method of any one of claims 1-12, wherein the conjugate moiety is conjugated to the acceptor glutamine residue by transglutaminase amidation.
14. The method of any one of claims 1-13, wherein said anti-HER 2 antibody comprises: a heavy chain variable region (VH) comprising: heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO. 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO. 3; and a light chain variable region (VL) comprising: light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO. 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO. 6.
15. The method of claim 14, wherein said anti-HER 2 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO. 7 and a VL comprising the amino acid sequence of SEQ ID NO. 8.
16. The method of any one of claims 1-15, wherein the Fc region is IgGl Fc.
17. The method of claim 16, wherein said anti-HER 2 antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO 9 and a light chain comprising the amino acid sequence of SEQ ID NO 10.
18. The method of any one of claims 1-17, wherein the toxin is monomethyl auristatin e (mmae).
19. The method of any one of claims 1-18, wherein the conjugate moiety comprises a cleavable linker.
20. The method of claim 19, wherein the conjugate moiety is a compound of formula I:
Figure DEST_PATH_IMAGE001
21. the method of any one of claims 1-20, wherein the antibody-drug conjugate is DP303 c.
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WO2023044016A2 (en) * 2021-09-17 2023-03-23 Cspc Dophen Corporation Antibody immune-stimulant conjugates and use thereof
WO2023070125A1 (en) * 2021-10-22 2023-04-27 Academia Sinica Antibody-drug conjugate for reducing glycosylation of membrane glycoprotein
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