CN111773385A

CN111773385A - Application of ErbB2 antibody and Saracatinib in preparation of drugs for treating breast cancer

Info

Publication number: CN111773385A
Application number: CN202010808800.1A
Authority: CN
Inventors: 彭菲; 顾超
Original assignee: Beijing Yuehao Technology Development Co ltd
Current assignee: Zhejiang shimai Pharmaceutical Co.,Ltd.
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-10-16
Anticipated expiration: 2040-08-12
Also published as: CN111773385B

Abstract

The invention relates to application of an ErbB2 antibody and Saracatinib in preparation of a drug for treating breast cancer, and the ErbB2 antibody and Saracatinib have the effect of synergistically inhibiting growth of an ErbB2 positive tumor and have wide application prospects when combined.

Description

Application of ErbB2 antibody and Saracatinib in preparation of drugs for treating breast cancer

Technical Field

The invention relates to the field of biological medicines, in particular to application of an anti-ErbB 2 antibody and Saracatinib in combination in treating breast cancer.

Technical Field

In recent years, the incidence of global clinical cancers is the first incidence of female malignant tumors, and the incidence of global cancers is second to lung cancer, so that the breast cancer is obviously the first major malignant tumor harmful to female health, the surgical operation is the main means for treating the breast cancer, and chemotherapy and radiotherapy are recognized auxiliary treatment methods. However, in recent years, with the continuous and deep research on targeted therapy and anti-tumor methods, the development of molecular pathology and genotyping results in the rapid development of monoclonal antibody drugs for breast cancer. Wherein trastuzumab ((trade name Herceptin, Herceptin) is the first antibody drug used for the treatment of ErbB2 positive metastatic breast cancer.

ErbB2, epidermal growth factor receptor 2 (also known as Neu, HER2, CD340 or p185), belongs to the epidermal growth factor receptor (EGFR/ErbB) family, is predominantly localized on the cell membrane and is expressed in the cytoplasm in small amounts. The outer membrane region comprises four structural domains of I, II, III and IV, and the multiple ring structures of the inner membrane region enable the inner membrane region to have tyrosine kinase activity. There are two active forms of ErbB 2: one is a ligand-independent homodimer, and the other is a ligand-dependent heterodimer, so that downstream related pathways mainly based on P13K-PKB/Akt and MAPK pathways are activated, and thus, the downstream related pathways have multi-effect influence on the proliferation, differentiation, migration, adhesion, transformation and survival of cells, wherein the heterodimer generates stronger signals and is the most important signal transduction pathway in the dimer. Clinical studies show that 25% -30% of breast cancer patients have the over-expression of the ErbB2 gene in the cancer tissues, and the breast cancer over-expressed by the ErbB2 has the advantages of high growth speed, strong invasiveness and easiness in early metastasis. It is not sensitive to radiotherapy and chemotherapy, and is easy to recur. Therefore, the disease-free life cycle is short and the prognosis is poor. The ErbB2 antibody can prevent homo/heterodimerization by acting on ErbB2 on the surface of a tumor, further block a P13K/AKT and MAPK signal pathway and/or can remarkably reduce the expression of ErbB2, and reverse the malignant phenotype of cells; and NK cells, macrophages, neutrophils and the like expressing IgGFc receptors are recruited to exert antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) so as to kill tumor cells, thereby achieving the effects of inhibiting proliferation and resisting survival.

The response rate of Herceptin single-drug therapy in metastatic breast cancer reaches 15-35%, and when the Herceptin single-drug therapy is combined with single-dose chemotherapy drugs, the response rate is increased to 50-80%. In the adjuvant treatment of ErbB2 positive breast cancer where lymph node metastasis has occurred or there is a high risk of lymph node metastasis, if herceptin is applied simultaneously or sequentially with chemotherapy, the recurrence rate of breast cancer can be reduced by around 50% and the overall survival rate can be increased by 33%. Although Herceptin has achieved great success in clinical treatment, only about 30% of breast cancer patients respond to initial treatment with Herceptin, and most of these initial treatments have developed tumor recurrence and progression in the next year after treatment for effective breast cancer. In recent years, much research has been focused on the mechanism by which herceptin resistance can occur. These include overexpression of the membrane-associated glycoprotein mucin 4(mucin4, MUC-4) molecule that blocks trastuzumab binding sites or directly activates intracellular tyrosine kinases of ErbB2, formation of ErbB2 splice mutants (p95-HER2) that can still heterodimerize with HER3 and activate downstream pathways, mutations in key molecules of signaling pathways (e.g., PTEN loss, AKT signaling enhancement, SRC family activation) that lead to constitutive activation of downstream signals, activation of other members of the HER family or other receptors that mediate reactivation of downstream signaling pathways (e.g., EGFR, IGF-1R, CXCR4, integrin β 1), anti-apoptotic proteins, and dysregulation of cell cycle inducing cell cycle arrest, among others. Researchers have found that not only can herceptin be further improved in antitumor activity when used in combination with certain drugs, but also the resistance of ErbB2 positive breast cancer cells to herceptin can be overcome to some extent.

Disclosure of Invention

Based on the above findings, the present invention provides a novel ErbB2 antibody that can inhibit the growth of ErbB2 positive cells or tumors, either alone or in synergy with other drugs.

The invention provides the following technical scheme:

a humanized anti-ErbB 2 antibody comprising a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region sequence is SEQ ID NO. 1 and the light chain variable region sequence is SEQ ID NO. 5.

The heavy and light chain variable regions of the ErbB2 antibody of the present invention each have 3 Complementarity Determining Regions (CDRs). The 3 CDR sequences of the heavy chain variable region of the ErbB2 antibody are respectively SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.

The heavy and light chains of the ErbB2 antibody of the invention further comprise constant regions, and the antibody light chain constant region comprises human kappa and lambda chain sequences, and more preferably a kappa chain. The antibody heavy chain constant region comprises human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM, and more preferably IgG 1.

In some embodiments, the ErbB2 antibody is capable of binding ErbB2 with high affinity, using Biacore analysis, the anti-ErbB 2 antibody has a K of 2.13nM or less_DBinds ErbB 2.

In some embodiments, the ErbB2 antibody is capable of inhibiting proliferation of SK-BR3 breast cancer cells highly expressed in ErbB 2.

In some embodiments, the ErbB2 antibody is capable of inhibiting heregulin-beta induced proliferation of MCF-7 breast cancer cells under-expressed ErbB 2.

The invention provides a method for treating breast cancer, which is characterized by comprising the following steps: patients with breast cancer using the anti-ErbB 2 antibody of the invention were ErbB2 positive.

The ErbB2 antibody can be applied to preparation of medicaments for treating and/or preventing breast cancer, and a breast cancer patient is positive to ErbB 2.

The combination methods of the invention may additionally comprise agents that inhibit the activation of the PI3K/AKT/mTOR pathway, small molecule tyrosine kinase inhibitors of the HER family, Src kinase inhibitors, and the like.

The drugs for inhibiting the activation of PI3K/AKT/mTOR pathway contained in the combined application method can include but are not limited to PI3K inhibitors GDC-0941 (Picilisib), NVP-BEZ23 (Dactlisib), mTOR inhibitors sirolimus (Temsirolimus, CCI-779), Lonafarnib (Lonafanib, SCH66336), Everolimus (RAD 001), Ridaoliformimus (Deformolimus, AP23573, MK-8669), AKT inhibitors MK-2206 and the like.

The small molecule tyrosine kinase inhibitor of HER family contained in the combined application method can include, but is not limited to, small molecule tyrosine kinase inhibitor Lapatinib (Lapatinib) reversibly binding to HER1 and ErbB2, small molecule tyrosine kinase inhibitor Gefitinib (Gefitinib, ZD1839) selectively binding to HER1, Erlotinib (Erlotinib), small molecule tyrosine kinase inhibitor canatinib (Canertinib, CI-1033) irreversibly inhibiting HER1, ErbB2, HER3 and HER4, small molecule tyrosine kinase inhibitor Afatinib (Afatinib, BIBW-2992) irreversibly binding to HER1 and ErbB2, small molecule tyrosine kinase inhibitor Neratinib (Neratatinib, HKI-272) irreversibly binding to HER1, ErbB2 and HER4, and the like.

The Src kinase inhibitors included in the combined application method of the present invention may include, but are not limited to, Saracatinib (AZD-0530), Bosutinib (SKI-606), KX1-004, and the like.

In a specific embodiment, the method of combined use for the treatment of HER2 positive breast cancer comprises an ErbB2 antibody consisting of the VH chain shown in SEQ ID NO:1 and the VL chain shown in SEQ ID NO:5 and the PI3K inhibitor GDC-0941 (Picilisib).

In a specific embodiment, a method of combination therapy for HER2 positive breast cancer comprises an ErbB2 antibody consisting of the VH chain shown in SEQ ID NO:1 and the VL chain shown in SEQ ID NO:5 and the small molecule tyrosine kinase inhibitor Neratinib (Neratinib, HKI-272).

In a specific embodiment, the method of combined use for the treatment of HER2 positive breast cancer comprises an ErbB2 antibody consisting of the VH chain shown in SEQ ID NO:1 and the VL chain shown in SEQ ID NO:5 and the Src kinase inhibitor Seatinib (Saracatinib, AZD-0530).

The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (antigen-binding portion) thereof or single chain homolog thereof. An "antibody" comprises at least one heavy (H) chain and one light (L) chain. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs), interspersed with more conserved regions known as Framework (FR) or junction (J) regions (JH or JL in heavy and light chains, respectively).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a plurality of substantially homologous antibody populations, i.e., the individual antibodies comprising the antibody population are identical except for mutations in naturally occurring cattle that may be present in minor amounts.

The term "polyclonal antibodies" as used herein refers to antibody compositions having a heterogeneous population of antibodies. Polyclonal antibodies are typically derived from collected sera of immunized animals or from selected humans.

The term "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. CDR region sequences can be defined by Kabat, Chothia method definition or the field of any known CDR region sequence determination method and identification of the variable region within amino acid residues. The methods used in the present invention may utilize or be defined according to CDRs defined by any of these methods, including but not limited to any of the Kabat definitions, Chothia definitions. In particular, the CDR sequences provided herein are according to the Kabat definition.

The term "humanized antibody" as used herein generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of the xenogenous antibody when introduced into humans, while maintaining the full antigen-binding affinity and specificity of the antibody. For example, CDR grafting and variants thereof can be used; the method comprises technical means such as remodeling, high-degree addition, veneering, surface reconstruction and the like, and humanizes the non-human-derived binding domain. Other regions, such as the hinge region and constant region domains, may also be humanized if they are also derived from non-human sources.

The term "K" as used herein_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction or the affinity of an antibody for an antigen. In one embodiment, an antibody according to the invention has an affinity (K) of 100nM or better_D) Binding to antigen, as measured using a surface plasmon resonance assay, a cell binding assay, or an equilibrium dialysis assay. In particular embodiments, the antibody binds ErbB2 with an affinity of 2.13nM or more, as measured by a surface plasmon resonance assay or a cell binding assay. In other embodiments, the antibody is less than about 10 when measured by Surface Plasmon Resonance (SPR) techniques in a BIACORE3000 instrument using recombinant ErbB2 as the analyte and an antibody as the ligand^-7M, e.g. about less than 10^-8M、10^-9M or even lower affinity binds to the antigen ErbB 2.

The term "synergistic effect" or "synergistic effect" as used herein is meant to encompass a combined therapeutic effect of two agents that is greater than the additive therapeutic effect of the two agents administered alone.

Advantageous effects

The invention has the following beneficial effects: the provided novel ErbB2 antibody can inhibit the growth of ErbB2 positive cells or tumors, and has the effect of synergistically inhibiting the growth of ErbB2 positive tumors when combined with Saracatinib.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 mouse antibody subtype identification

FIG. 2 anti-ErbB 2 antibody Hu3-7B4 inhibits SK-BR3 cell proliferation

FIG. 3 anti-ErbB 2 antibody Hu3-7B4 inhibits heregulin beta-induced MCF-7 cell proliferation

FIG. 4 shows that the combination of anti-ErbB 2 antibody Hu3-7B4 and Pictilisib has the growth inhibition effect on transplanted tumor in nude mice model with breast cancer

FIG. 5 growth inhibition of anti-ErbB 2 antibody Hu3-7B4 and Neratinib on transplanted tumor in nude mice model of breast cancer

FIG. 6 shows that the combination of anti-ErbB 2 antibody Hu3-7B4 and Saracatinib has the growth inhibition effect on transplanted tumor in nude mice model with breast cancer

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 anti-ErbB 2 antibody preparation and purification

Balb/c healthy female mice homologous to SP2/0 were selected as immunized animals using recombinant human ErbB2 extracellular domain (ErbB2-ECD) protein as immunogen. Dissolving the immune antigen in PBS, and uniformly mixing with Freund's adjuvant with the same volume to form the water-in-oil emulsion. The primary immunization was performed by subcutaneous multiple immunization at a dose of 100. mu.g/mouse. 7-10 days after the initial immunization, mice were immunized in the same way and at the same dose by mixing and emulsifying incomplete Freund's adjuvant and immunizing antigen in equal volume. Then boosting the immunity for several times every 7-10 days to make the antibody titer of the mouse serum reach more than ten thousand. One week after the last booster immunization, an eye bleed was performed and the serum titer was determined by ELISA. If the serum titer of the mice does not reach ten thousand, the boosting is required to be continuously performed for several times. Three days before fusion, the mice with the potency of more than ten thousand are subjected to impact immunization, and the tail vein is injected with antigen without adjuvantTaking immunized Balb/c mouse spleen cells, fusing the immunized Balb/c mouse spleen cells with a myeloma SP2/0 cell line by a PEG method, wherein the number ratio of SP2/0 cells to spleen cells is usually 1: 3-1: 5, adding HAT (Hatch-associated protein) into the fused cells, adding cell suspension into a plurality of 96-well cell culture plates according to 100 mu l/well, and ensuring that the cell amount of each well is about 4 × 10⁴Each cell/well was cultured in a 37 ℃ cell incubator. On day four of confluency, HAT medium was added to continue the culture. After about 12 days of cell fusion, the culture supernatant was aspirated, and positive hybridoma cell lines were screened by indirect ELISA. Screening monoclonal positive hybridoma cell strain by limiting dilution method, enlarging culture of positive hybridoma cell, and freezing for seed preservation. The finally screened positive hybridoma cell line was named 3-7B 4.

Example 2 identification of mouse anti-ErbB 2 antibody subtypes

The anti-ErbB 2 monoclonal antibody obtained in example 1 was identified by ELISA (kit from Proteitech). The microplate provided in the kit was already pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-NSE antibody samples E1-7E2 and E2-4F6 purified in example 2 were added to sample wells, 50. mu.l per well, respectively, without incubation. Adding 1X goat anti-mouse IgA + IgM + IgG-HRP into sample wells, mixing the sample wells with 50 μ l each, and incubating for 1 h. And (4) deducting liquid in the holes, adding 1XPBST to wash the holes for 3 times, and absorbing the excessive moisture by absorbent paper. Adding color development solution, and developing 100 μ l per well in dark at room temperature for 15 min. The color reaction was stopped by adding 100. mu.l of stop solution. The OD value at 450nm is detected by a microplate reader, and the result is shown in figure 1, the heavy chain subtypes and the light chain subtypes of the anti-ErbB 2 monoclonal antibody 3-7B4 are all IgG1 and Kappa respectively.

Example 3 antibody humanization

The positive hybridoma cells obtained in example 1 were expanded and a suitable amount of the cells were collected and used as TRNzol-A⁺After lysis, 1ml of TRNzol-A per unit⁺Add 200. mu.l chloroform, vortex for 15 seconds, and let stand for 3 minutes. 13000rpm, centrifugation at 4 ℃ for 10 minutes, the cell solution was divided into three layers: the upper colorless aqueous phase, the middle layer and the lower yellow organic phase, transferring the aqueous phase dissolved with RNA to centrifugationIn a tube, equal volume of isopropanol was added to the aqueous phase, mixed well and allowed to stand at room temperature for 25 minutes. 13000rpm, 4 ℃ centrifugal 10 minutes, discarded waste liquid to get the bottom of the RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in DEPC water. First strand cDNA was then synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANS, AT311-02) reverse transcription kit. Specific primers (the 5' end contains a homologous arm sequence for homologous recombination with a eukaryotic expression vector) are designed according to the identification result of the mouse antibody subtype in the example 2, and the cDNA is used as a template to perform PCR amplification of antibody variable region genes, so that gene fragments of mouse antibody light chain and heavy chain variable regions are respectively obtained. Humanization of antibodies was first performed by aligning the amino acid sequences of the VH and VK domains of the antibody with the amino acid sequences of all known human germline VH and VK domains using currently available public databases (i.e., Blast for IgG from NCBI and V-base from MRC). By observing the alignment of amino acids within the framework regions, highly homologous human germline VH and VK domains are determined. On the basis of the framework structure, the computer model is used for analyzing the variable region steric structure of the mouse anti-ErbB 2 antibody 3-7B4, and the corresponding framework amino acid residues of the mouse antibody required to be reserved for supporting the CDR configuration are analyzed. The mouse frameworks are transformed or mutated in an iterative manner to match the corresponding human germline frameworks. The synthetic VH and VK domains were cloned into a specialized mammalian expression vector that enabled expression of the respective domains in the complete human IgG1 or kappa antibody backbone. The lgG 1 construct was co-transfected with the kappa construct into 293F cells for small-scale preparation of humanized antibodies. The supernatant of the transient transfection was passed through protein a or G resin to obtain purified ErbB2 antibody. Finally, the humanized and transformed antibody is named Hu3-7B4, the VH chain amino acid sequence of the antibody Hu3-7B4 is shown as SEQ ID NO. 1, and the VL chain amino acid sequence is shown as SEQ ID NO. 5; wherein the amino acid sequences of the heavy chain hypervariable region VH-CDR1, VH-CDR2 and VH-CDR3 are respectively shown as SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4; the amino acid sequences of the light chain hypervariable regions VL-CDR1, VL-CDR2 and VL-CDR3 are respectively shown as SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8。

Example 4 affinity assay for humanized antibodies

Coating a human Fc antibody capture Antibody (AHC) on the surface of a CM5 chip by means of amino coupling, preparing chip activation buffer EDC/NHS, AHC and ethanolamine for blocking by referring to the specifications of an amino coupling kit and a human anti-capture kit, selecting a coating program in a Biacore3000 system, and coupling the AHC amino on the surface of a CM5 chip. The humanized ErbB2 antibody Hu3-7B4 antibody obtained in example 2 was captured on the surface of the chip. The antibody was diluted to 2. mu.g/mL with HBS-EP + buffer, set to a flow rate of 10. mu.L/min, and coated to a response value of 300 RU. The ErbB2-ECD antigen was subjected to 7 different concentration gradients, which were serially diluted 2-fold in HBS-EP buffer at concentrations ranging from 200nM to 3.125 nM. The flow rate of the ErbB2-ECD antigen was set at 50. mu.L/min and the binding time was set at3 min. The flow rate of HBS-EP + buffer is set to be 50 mu L/min, and the dissociation time is set to be 10 min. Using 3MMgCl₂As a regeneration buffer, the chip was regenerated according to the regeneration procedure. Affinity assays were performed using humanized anti-human HER2/ErbB2 humanized anti-Herceptin as a positive control (PDB: 1N8Z for sequence reference) against the antibody following the same procedure. The results are shown in table one: the affinity of the antibody of the invention is obviously superior to that of a control antibody Herceptin.

Watch 1

Antibodies	Kon(1/Ms)	Kdis(1/s)	K_D(nM)
				Hu3-7B4	1.16x10⁵	2.47x10^-5	2.13
Herceptin	2.16x10⁵	9.72x10^-5	4.50

Example 5 anti-ErbB 2 antibody Hu3-7B4 inhibits SK-BR3 cell proliferation

The effect of the anti-ErbB 2 antibody Hu3-7B4 obtained in example 2 on the proliferation of SK-BR3 cells was determined using the MTT assay. After digesting a good-state and high-expression breast cancer cell line SK-BR3 of ErbB2 in logarithmic growth phase, respectively inoculating 4000-6000 cells in a 96-well plate, respectively adding anti-ErbB 2 monoclonal antibody Hu3-7B4, positive control Herceptin or negative control IgG1 with concentration gradients of 5 mug/mL, 10 mug/mL and 20 mug/mL the next day, wherein each well is filled with l00 mug, and each concentration is provided with 3 repeated wells. Blank control wells were also set, i.e., complete medium without antibody was added. Incubation at 37 ℃ was carried out during which the state of the cells under different concentration treatment conditions was observed in an inverted microscope at 24h intervals. 5% CO₂After incubation at 37 ℃ for 72 hours, 100. mu.l of MTT solution (5mg/mL) was added to each well, and after 4 hours, the culture was terminated and the culture solution in the wells was carefully aspirated. Add 100 μ l dimethyl sulfoxide into each well, shake for 10min at low speed on the shaking table to dissolve the crystals completely. And detecting the absorbance value of each hole by a microplate reader at 570 nm. The result is shown in figure 2, the anti-ErbB 2 antibodies Hu3-7B4 and Herceptin have obvious growth inhibition effect on the breast cancer cell strain SK-BR3 with high expression of ErbB2, which shows that Hu3-7B4 can inhibit the growth of breast cancer cells and the biological activity is not weaker than that of positive control Herceptin.

Example 6 anti-ErbB 2 antibody Hu3-7B4 inhibits heregulin beta-induced MCF-7 cell proliferation

MTT experiment is utilized to determine the influence of the anti-ErbB 2 antibody Hu3-7B4 obtained in example 2 on the MCF-7 cell proliferation induced by the regulatory protein β. the breast cancer cell strain MCF-7 which is in a good state and is expressed at a low level by ErbB2 in logarithmic growth phase is eliminatedAfter the cell transformation, 4000-6000 cells per well are respectively inoculated on a 96-well plate, 50 mu l of heregulin β of 2nM is added into each well the next day, anti-ErbB 2 monoclonal antibody Hu3-7B4, positive control Herceptin or negative control IgG1 with concentration gradient of 2.5 mu g/mL, 5 mu g/mL and 10 mu g/mL are respectively added into an experimental well, l00 mu l of each well is provided with 3 repeated wells for each concentration, blank control wells are simultaneously arranged, namely complete culture medium without antibody is added, the incubation is carried out at 37 ℃, the cell state under the condition of different concentrations of treatment is observed in an inverted microscope at intervals of 24h, and 5% CO is used for observing the cell state under the condition of different concentrations₂After incubation for 72h at 37 ℃, 100 mu l of MTT solution (5mg/mL) is added into each hole, the culture is stopped after 4h, culture solution in each hole is carefully absorbed, 100 mu l of dimethyl sulfoxide is added into each hole, the shaking table is shaken at low speed for 10min, crystals are fully dissolved, an enzyme-labeling instrument is used for detecting the absorbance value of each hole at 570nm, the result is shown in figure 3, an anti-ErbB 2 antibody Hu3-7B4 has obvious growth inhibition effect on a breast cancer cell line MCF-7 with low-level expression of ErbB2 induced by opsin β, while Herceptin has no obvious growth inhibition effect on the breast cancer cell line MCF-7, Hu3-7B4 has obvious inhibition effect on the growth of the MCF-7 cells induced by opsin β, and the biological activity is obviously superior to that of Herceptin.

Example 7 growth inhibition of the combination of ErbB2 monoclonal antibody Hu3-7B4 and Pictilisib on model transplantable tumors in nude mice with Breast cancer

To investigate the effect of the ErbB2 monoclonal antibody Hu3-7B4 in combination with PI3K inhibitor Pictilinib on the growth of breast cancer, a BT474 breast cancer xenograft tumor model was constructed in 6-8 week-old female NOD/SCID mice, sufficient breast cancer cell line BT-474 tumor cells highly expressing ErbB2 in logarithmic growth phase were cultured, the cells were collected and washed with PBS, and then the cell concentration was diluted to an appropriate concentration.BT-474 cells were mixed with a basement membrane (Matrigel; collagen Research, Bedford, MA) at a volume ratio of 1:1 and inoculated under the breast pad of mice, and each mouse was inoculated with 1 × 10⁷And BT-474 cells. Until the BT474 breast cancer transplantation tumor grows to about 150m³At the time, we randomly divided tumor-bearing nude mice into 4 groups of 6 mice each and administered the experiments, including: (a) group 1: PBS (vehicle control); (b) group 2: only the ErbB2 antibody Hu3-7B4,5mg/kg, (c) group 3, 100mg/kg of PlI 3K inhibitor Pictilisb alone, (d) group 4, Hu3-7B4+ Pictilisb, wherein the ErbB2 antibody Hu3-7B4, 5mg/kg, PI3K inhibitor Pictilisb, 100 mg/kg., were administered by intravenous injection of Hu3-7B4 weekly tail edges, by daily gavage, tumor length, short diameter, and tumor size were calculated by the formula of tumor volume implanted, tumor long diameter × tumor wide diameter × tumor wide diameter ×.5. when the entire experiment reached the observation end, nude mice were sacrificed by cervical dislocation.

Example 8 growth inhibition of ErbB2 monoclonal antibody Hu3-7B4 in combination with Neratinib on transplanted tumors in nude mice model of breast cancer

To investigate the effect of Hu3-7B4 in combination with the small molecule tyrosine kinase inhibitor Neratinib on the growth of breast cancer, 6-8 week-old female NOD/SCID mice were used to construct a BT474 breast cancer xenograft tumor model, enough ErbB 2-highly expressed breast cancer cell line BT-474 tumor cells in logarithmic growth phase were cultured, the cells were collected and washed with PBS, and then the cell concentration was diluted to an appropriate concentration, BT-474 cells were mixed with basement membrane (Matrigel; collagen research, Bedford, MA) at a volume ratio of 1:1, and then inoculated under the breast pad of mice, and each mouse was inoculated with 1 × 10⁷And BT-474 cells. Until the BT474 breast cancer transplantation tumor grows to about 150m³At the time, we randomly divided tumor-bearing nude mice into 4 groups of 6 mice each and administered the experiments, including: (a) group 1: PBS (vehicle control); (b) group 2: only the ErbB2 antibody Hu3-7B4, 5 mg/kg; (c) group 3: only small molecule tyrosine kinase inhibitor Neratinib, 10 mg/kg; (d) group 4: hu3-7B4+ Neratinib, wherein the ErbB2 antibody Hu3-7B4, 10mg/kg and the small molecule tyrosine kinase inhibitor Neratinib, 5 mg/kg. The administration route isHu3-7B4 is injected by tail vein every week, Neratinib is administrated by intragastric administration every day, tumor length and short diameter are measured every 3 days, and tumor size is calculated according to the formula that the volume of transplanted tumor is equal to the tumor width of × tumor with tumor length of × tumor, the tumor width is ×.5, when the whole experiment reaches the observation end point, nude mice are killed by cervical dislocation method, the experimental result is shown in figure 5, the tumor volume of nude mice in a negative control group is mostly in progressive growth, the effect of Neratinib alone and the effect of Hu3-7B4 alone both can inhibit the tumor growth to a certain extent, and the effect of Neratinib alone is obviously better than the effect of Hu3-7 B4. in combination of Hu3-7B4 and Neratinib, the tumor growth inhibition effect is only slightly changed, the combined effect does not achieve the additive and therapeutic effects, so that Hu3-7B4 and Neratinib have no synergistic effect.

Example 9 growth inhibition of ErbB2 monoclonal antibody Hu3-7B4 in combination with Saracatinib on transplanted tumors in nude mice model of Breast cancer

To investigate the effect of Hu3-7B4 in combination with Src kinase inhibitor Saracatinib on the growth of breast cancer, 6-8 week-old female NOD/SCID mice were used to construct a BT474 breast cancer xenograft tumor model, enough ErbB 2-highly expressed breast cancer cell line BT-474 tumor cells in logarithmic growth phase were cultured, the cells were collected and washed with PBS, and then the cell concentration was diluted to an appropriate concentration, BT-474 cells were mixed with basement membrane (Matrigel; collagen research, Bedford, Mass.) at a volume ratio of 1:1, and then inoculated under the breast pad of mice, and each mouse was inoculated with 1 × 10⁷And BT-474 cells. Until the BT474 breast cancer transplantation tumor grows to about 150m³At the time, we randomly divided tumor-bearing nude mice into 4 groups of 6 mice each and administered the experiments, including: (a) group 1: PBS (vehicle control); (b) group 2: only the ErbB2 antibody Hu3-7B4, 5 mg/kg; (c) group 3: src kinase inhibitor Saracatinib only, 25 mg/kg; (d) group 4: hu3-7B4+ Saracatinib, wherein ErbB2 antibody Hu3-7B4, 5mg/kg, Src kinase inhibitor Saracatinib, 25 mg/kg. The routes of administration are: hu3-7B4 was injected intravenously at the trailing edge of the week; saracatinib was administered by daily gavage. Measuring the length and the short diameter of the tumor every 3 days, calculating the size of the tumor, and calculatingThe formula is that the volume of transplanted tumor is equal to the wide diameter of tumor, ×, ×, × 0.5.5, when the whole experiment reaches the observation end point, nude mice are killed by cervical dislocation, the experimental result is shown in figure 6, the tumor volume of nude mice of a negative control group is mostly in progressive growth, Hu3-7B4 alone can inhibit the tumor growth to a certain extent, but Saracatinib alone has weak tumor inhibition effect, but the surprising finding shows that the combination of Hu3-7B4 and Saracatinib obviously enhances the tumor growth inhibition effect and the effect is obviously superior to the addition and treatment effect of the two, so the Hu3-7B4 and Saracatinib have good synergistic effect.

Sequence listing

<110> Beijing Yuehao science and technology development Co., Ltd

Application of <120> ErbB2 antibody and Saracatinib in preparation of drugs for treating breast cancer

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>118

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Thr Ser Ala

20 25 30

Lys Asn Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

3540 45

Ala Ile Trp Glu Asp Cys Lys Ala Arg Ser Gly Asp Ile Tyr Phe Ser

50 55 60

Lys Gly Ile Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Thr Gln Met Asn Ser Thr Ile Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Ile Asn Ser Asp Val Ala Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>2

<211>5

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Ser Ala Lys Asn Gly

1 5

<210>3

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Ile Trp Glu Asp Cys Lys Ala Arg Ser Gly Asp Ile Tyr Phe Ser Lys

1 5 10 15

Gly

<210>4

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Tyr Ile Asn Ser Asp Val Ala Asp Tyr

1 5

<210>5

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Cys Ile Asp Gly Ser Thr Lys

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ile Ala Asn Lys Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asp Asn Thr Ala Cys Gly Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>6

<211>11

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Arg Ala Cys Ile Asp Gly Ser Thr Lys Val Ala

1 5 10

<210>7

<211>7

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>7

Ser Ile Ala Asn Lys Tyr Ser

1 5

<210>8

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Gln Asp Asn Thr Ala Cys Gly Pro Thr

1 5

Claims

1. A pharmaceutical combination comprising: a) an anti-ErbB 2 antibody, consisting of a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence VH-CDR1 as shown in SEQ ID NO. 2, and the amino acid sequence VH-CDR2 as shown in SEQ ID NO. 3, and the amino acid sequence VH-CDR3 as shown in SEQ ID NO. 4; the light chain comprises a VL-CDR1 amino acid sequence shown as SEQ ID NO. 6, and a VL-CDR2 amino acid sequence shown as SEQ ID NO. 7, and a VL-CDR3 amino acid sequence shown as SEQ ID NO. 8; and b) the Src kinase inhibitor Saracatinib.

2. The pharmaceutical combination of claim 1, wherein the ErbB2 antibody of claim 1 comprises the heavy chain variable region set forth in SEQ id no: 1; the light chain comprises a light chain variable region as shown in SEQ ID No. 5.

3. Use of a pharmaceutical combination according to any one of claims 1 to 2 for the treatment of ErbB2 positive breast cancer.