A mutant of an anti-cd38 antibody

Abstract

This invention discloses an anti-CD38 antibody mutant, relating to the field of biomedical technology. This invention is based on the modification of the Isatuximab antibody, aiming to retain its inhibitory effect on CD38 / NADase while eliminating its immune-activating effect. Specifically, this invention modifies antibody V... H Mutations of Y27H, G99A, and N105D were performed in the region, and mutations of L237A, L238E, and P332G were performed in the Fc region, successfully obtaining the modified antibody mutant. The modified antibody showed a stronger inhibitory effect on CD38 / NADase activity than the original Isatuximab, while retaining comparable affinity and specificity to Isatuximab.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to anti-CD38 antibody mutants and their applications. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) + NAD+ is an important intracellular coenzyme that participates extensively in various cellular biochemical reactions. Studies have shown that NAD+... + It plays an important protective role in many diseases. Abnormal NAD + Levels of NAD+ have been shown to be closely related to the occurrence and progression of neurodegenerative diseases, aging, pulmonary fibrosis, and other conditions. Supplementation with exogenous NAD+ can help. + Or inhibit NAD in the body + The degradation of NAD+ is believed to help improve these diseases. Currently, NAD+... + Its precursor compounds have been widely promoted as health supplements, and can promote cellular energy metabolism, anti-aging, and improve metabolic health.

[0003] CD38 is a single-chain type II transmembrane glycoprotein with a unique enzymatic active site, shaped like a "pocket," which enables it to effectively recognize and bind to NAD. + CD38 regulates intracellular calcium... 2+ Signaling and intracellular NAD + Concentration affects cellular energy state and is the main NAD+ concentration in mammalian cells. + CD38 is a degradation enzyme and participates in the regulation of the aging process. Therefore, CD38 and NAD... + Metabolic interactions offer potential targets for developing novel therapeutic strategies, particularly targeting NAD. + It has important application value in the treatment of related diseases.

[0004] Targeting and blocking CD38's effect on NAD + Its degradation function is a way to enhance the body's NAD. + Levels, improve NAD + Potential treatment strategies for related diseases. Currently, there are two main approaches to drug development that blocks CD38 enzyme activity: small molecule competitive inhibitors and specific antibody blockade. Small molecule inhibitors have advantages such as high permeability, good bioavailability, and low cost, but their disadvantages include poor specificity, potential adverse reactions, and a short half-life, requiring frequent dosing. Specific antibody drugs, on the other hand, exhibit high specificity against CD38, have fewer side effects, and have a longer half-life, effectively reducing the frequency of dosing and making them suitable for long-term treatment.

[0005] Currently, CD38 monoclonal antibodies are available for clinical treatment, such as Daratumumab and Isatuximab. These antibodies are primarily used for immunotherapy of multiple myeloma (MM) but are not NAD-dependent. + Metabolic mechanisms. Antibody research targeting CD38 enzyme activity typically employs two strategies: one is to block the CD38 enzyme activity pocket with antibodies, and the other is to indirectly block NAD by altering the spatial conformation of CD38 after antibody binding. + The combination of CD38 and NAD. + Due to the unique structure of the binding domain, it is almost impossible for the innate immune system to generate antibodies against this region.

[0006] Isatuximab is a humanized IgG1 monoclonal anti-CD38 antibody approved by the FDA and EMA for the treatment of relapsed / refractory multiple myeloma (RRMM). This antibody primarily works by specifically binding to different conformational epitopes of CD38 on the surface of tumor cells via its Fab region, and by mediating antibody-dependent cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP) through its Fc region. This activates effector cells such as NK cells and macrophages to clear tumor cells, initiating a multi-pronged anti-tumor mechanism.

[0007] Isatuximab has the ability to inhibit CD38 / NADase (NAD) + The unique function of CD38's NADase activity (hydrolyzing enzymes). CD38's NADase activity leads to NAD... + It degrades, generating ADPR and adenosine, thereby inhibiting the function of immune cells and negatively regulating the tumor immune microenvironment. Studies have shown that Isatuximab inhibits the enzymatic catalytic activity of CD38 and blocks NAD through allosteric antagonism. + The consumption of NAD may restore + It improves T cell function and enhances anti-tumor immune response.

[0008] However, due to the unique conformation of the CD38 enzyme's active site, it is difficult to obtain specific antibodies against the CD38 enzyme's active site through traditional animal immunization methods. Therefore, modifying the structure of Isatuximab to obtain more potent and specific anti-CD38 antibody mutants has become an ideal research direction. Summary of the Invention

[0009] This invention provides an anti-CD38 antibody mutant. This anti-CD38 antibody mutant, based on point mutation of wild-type Isatuximab, can target CD38, enhance CD38 / NADase inhibition, and regulate cellular NAD. +Levels, thus providing a basis for treatment and NAD + Metabolic-related diseases offer potential treatment strategies. The technical solution of this invention is achieved through the following technical means.

[0010] In a first aspect, the present invention provides an anti-CD38 antibody mutant, wherein the amino acid sequence of the anti-CD38 antibody mutant is based on the wild-type antibody Isatuximab, and its heavy chain is mutated by Y27H, G99A, N105D, L237A, L238E and P332G.

[0011] Specifically, the amino acid sequence of the heavy chain variable region of the wild-type antibody Isatuximab is shown in SEQ ID NO.1. The amino acid sequence of the heavy chain variable region of the anti-CD38 antibody mutant is shown in SEQ ID NO.2.

[0012] The mutants modified by the above point mutations exhibit stronger CD38 / NADase inhibitory activity, while retaining affinity and specificity comparable to Isatuximab.

[0013] Furthermore, the amino acid sequence of the heavy chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.4, and the amino acid sequence of the light chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.5.

[0014] Furthermore, the gene sequence encoding the heavy chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.8, and the gene sequence encoding the light chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.9.

[0015] In a second aspect, the present invention provides a nucleic acid molecule that encodes the amino acid sequence of the aforementioned anti-CD38 antibody mutant.

[0016] In a third aspect, the present invention provides a recombinant expression vector comprising the above-described nucleic acid molecule, wherein the recombinant expression vector is used to express the anti-CD38 antibody mutant.

[0017] In a fourth aspect, the present invention also provides a host cell containing the above-described recombinant expression vector.

[0018] A fifth aspect of the invention also provides a method for blocking NAD. + The consumed blocking agents include the aforementioned anti-CD38 antibody mutant; these blocking agents inhibit the enzymatic catalytic activity of CD38 by specifically binding to CD38, thereby blocking NAD. + The consumption of NAD may improve the relationship with NAD. + Metabolic diseases.

[0019] Furthermore, blocking NAD + The blocking agents consumed also include buffer solutions.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] 1. Enhanced inhibitory effect. This invention, based on the existing antibody Isatuximab, modifies it through point mutation to prepare antibody mutants that do not induce an immune response. These mutants can more effectively target and inhibit CD38 / NADase activity, thereby significantly improving the inhibitory effect on CD38 / NADase and exhibiting a stronger inhibitory effect than the original Isatuximab.

[0022] 2. Balancing safety and efficacy. This invention, through structural modification, avoids the immunotoxicity issues of Isatuximab while enhancing its inhibitory effect on CD38, resulting in lower side effects and better meeting clinical treatment needs.

[0023] 3. Broad application prospects. The anti-CD38 antibody mutant of this invention can theoretically be applied to NAD+ antibodies. + Potential treatments for metabolic disorders, particularly immune-related diseases, aging, and cancer. However, further experiments and validation are needed for practical clinical application. Attached Figure Description

[0024] Figure 1 Simulate Isatuximab and Isatuximab V for AlphaFold 3 H The effect of mutants on the conformation of huCD38.

[0025] Figure 2 The QC diagram of the final purified protein sample (Coomassie Brilliant Blue staining) is shown in the figure. A represents reducing SDS-PAGE, and B represents non-reducing SDS-PAGE.

[0026] Figure 3 The ε-NAD assay was used to detect the inhibitory effect of NCU-106 on CD38 / NADase activity in cells. In the figure, A and B are enzyme activity assays performed using two different cell lines, respectively.

[0027] Figure 4 For the detection of affinity and specificity of NCU-106.

[0028] Figure 5 To evaluate the ADCC effect of the NCU-106 antibody, A and B in the figure show ADCC detection using two different target cells. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Design of anti-CD38 antibody mutants

[0031] 1. Mechanism of Isatuximab-induced local conformational changes in huCD38 structure

[0032] It is known that Isatuximab (Isa) can induce a local conformational change in the huCD38 protein upon binding. Specifically, the Q115-V117 segment shifts approximately 2.5 Å toward the light chain, thereby creating sufficient space to accommodate the H3 ring of Fab.

[0033] To further explore their interaction mechanism, this embodiment used AlphaFold 3 to simulate the interaction between Isatuximab and huCD38 protein. Simulation results (see...) Figure 1 Figure B shows that the interaction simulation between the two has high confidence (ipTM=0.89). This is similar to the interaction with normal huCD38 protein (see Figure B). Figure 1 Compared to A), after Isatuximab binding, the M110-C118 segment of the huCD38 protein underwent a significant shift.

[0034] The above results are consistent with protein crystallographic data (Protein Bank ID: 4CMH), indicating that the AlphaFold 3 simulation accurately reflects the conformational changes of the huCD38 protein after Isatuximab binding. Therefore, based on the AlphaFold 3 simulation results, it is feasible to design an Isatuximab mutant that retains CD38 / NADase inhibitory activity.

[0035] 2. Anti-CD38 antibody mutation design principles

[0036] (1) Keep the Isatuximab antibody backbone unchanged, that is, do not change the FR (framework) sequence;

[0037] (2) For the variable region V of the Isatuximab heavy chain H Point mutations were designed in the three CDR sequences of the region;

[0038] (3) The amino acids in the CDR region of the point mutation should ensure that the AlphaFold simulation results do not change the effect of Isatuximab on the shift of the M110-C118 segment of the huCD38 protein, and the ipTM value should be ≥0.80;

[0039] (4) The number of amino acids in point mutations does not exceed 5.

[0040] Based on the above mutation design principles, a large-scale AlphaFold 3 simulation analysis was conducted, and the ideal mutation combination was finally selected: Y27H / G99A / N105D. The AlphaFold 3 simulation results are as follows: Figure 1 As shown in C, the mutant combination Y27H / G99A / N105D has a significantly stronger effect on the shift of the M110→C118 segment of the huCD38 protein than the wild-type Isatuximab, suggesting that this mutant combination may exhibit stronger CD38 / NADase inhibitory activity.

[0041] Heavy chain variable region V of wild-type Isatuximab H The amino acid sequence is shown in SEQ ID NO.1. The heavy chain variable region V of the Isatuximab mutant... H The amino acid sequence is shown in SEQ ID NO.2.

[0042] Example 2: Design of Fc region mutations in anti-CD38 antibody

[0043] To preserve or enhance the inhibitory activity of Isatuximab antibodies against CD38 / NADase while eliminating their immunotoxicity, it is crucial to understand that Isatuximab antibodies induce human immune cells (such as NK cells and macrophages) to recognize and kill labeled target cells via their Fc region. Therefore, strategies to eliminate the immunotoxicity of Isatuximab antibodies can be categorized into two types: one is to remove the Fc region, retaining only the Fab region; the other is to modify the Fc region through mutation design, rendering it unable to activate immunotoxicity.

[0044] Intact IgG antibodies offer significant advantages over incomplete antibody fragments such as Fab in terms of production efficiency, stability, and in vivo half-life. Therefore, in practical antibody drug design, engineered mutations are typically used to remove the immune effector function of the Fc region, thereby achieving a "pure blocking" effect or reducing immune-related toxicity.

[0045] The amino acid sequence of the Fc region of the human wild-type IgG1 antibody used in this invention is shown in SEQ ID NO.3. Based on existing literature, the Fc mutation combinations shown in Table 1 below were compiled, and the interaction between the Fc mutants and the high-affinity receptor huCD64 was simulated using AlphaFold 3. The mutation combinations were ranked according to their ipTM values; the lower the ipTM value, the less likely Fc binding to huCD64 will occur, thus providing a preliminary indication of the degree of immune activation.

[0046] Table 1 Evaluation of the binding ability of different mutation combinations in the Fc region of AlphaFold to huCD64

[0047]

[0048] As shown in Table 1, the final selected mutation combination was L237A / L238E / P332G, which had the lowest ipTM value.

[0049] Example 3: Complete sequence of anti-CD38 antibody mutant

[0050] In this embodiment, the heavy chain constant region C of Isatuximab H1 Light chain variable region V L and light chain constant region C L No mutations were performed. Combining the mutation combinations from Examples 1 and 2, the final antibody mutant was obtained and named NCU-106.

[0051] NCU-106's "V" H +C H The amino acid sequence of the region (heavy chain) is shown in SEQ ID NO.4, and the "V" of NCU-106 L +C L The amino acid sequence of the region (light chain) is shown in SEQ ID NO.5.

[0052] Example 4: Expression and purification of anti-CD38 antibody mutant NCU-106

[0053] The anti-CD38 antibody mutant NCU-106 was recombinantly expressed using a eukaryotic cell expression system. Specific steps included gene synthesis, plasmid (pATX1) construction, eukaryotic cell (XtenCHO cell) expression, and antibody purification (affinity chromatography purification) by Pujian Biotechnology (Wuhan) Co., Ltd.

[0054] In this embodiment, to obtain the anti-CD38 antibody mutant NCU-106, a secretory peptide was also attached. Part of the secretory peptide was attached to the heavy chain of NCU-106, and another part was attached to the light chain of NCU-106. The amino acid sequence of the secretory peptide attached to the NCU-106 heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the secretory peptide attached to the NCU-106 light chain is shown in SEQ ID NO.7. The V of NCU-106... H The gene sequence is shown in SEQ ID NO.8, V L The gene sequence is shown in SEQ ID NO. 9.

[0055] The heavy and light chain genes of the anti-CD38 antibody mutant NCU-106 were co-transfected into XtenCHO cells. On day 8 post-transfection, the culture supernatant was collected for purification of the anti-CD38 antibody mutant NCU-106.

[0056] The results of the purification process are shown in Figure 2 As can be seen, 2.10 mg of anti-CD38 antibody mutant NCU-106 protein was successfully purified from 100 mL of XtenCHO cell culture medium, with an antibody yield of 0.021 mg / mL. After thawing the protein solution stored at -80℃, it was incubated at 4℃ for 0.5 hours without any abnormalities, indicating that the protein freeze-thaw experiment results were normal.

[0057] Example 5: Detection of the inhibitory effect of NCU-106 antibody on CD38 / NADase activity

[0058] To verify whether the designed anti-CD38 antibody mutant NCU-106 retains its inhibitory function against CD38 / NADase, this experiment used nicotinamide 1,N6-vinyl adenine dinucleotide (ε-NAD) as a fluorescent substrate for enzyme activity assessment. ε-NAD contains a vinyl substituent in its structure, exhibiting fluorescence properties, and the degradation rate of ε-NAD by CD38 is significantly slower than that of NAD (approximately 5-7 times), providing a longer detection time window.

[0059] For cell selection, the cell lines RPMI-8226 (human multiple myeloma peripheral blood B lymphocytes) and Raji (human Burkitt's lymphoma cells), which highly express huCD38, were selected, both of which are suspension cells.

[0060] The experimental design includes the following four groups:

[0061] 1. Cell + ε-NAD group: Viable cells were seeded into 96-well plates (black sidewalls + clear bottom) (1×10⁶ cells / wells). 5(1 cell / well), then add ε-NAD to a final concentration of 50 μM.

[0062] 2. 78c group: 78c is a known small molecule inhibitor of CD38 / NADase enzyme. Cells with good activity were seeded into 96-well plates (black sidewalls + clear bottom) (1×10⁻⁶). 5 (1 cell / well), then add 78c to a final concentration of 200 nM, and add ε-NAD to a final concentration of 50 μM for enzymatic reaction.

[0063] 3. Isatuximab group: Viable cells were seeded into 96-well plates (black sidewalls + clear bottom) (1×10⁶ cells / wells). 5 (1 cell / well), then add Isatuximab antibody (two concentration gradients of 1 and 1000 ng / mL), pre-incubate at 37°C for 15 minutes, and then add ε-NAD to a final concentration of 50 μM for enzymatic reaction.

[0064] 4. NCU-106 group: Viable cells were seeded into 96-well plates (black sidewalls + clear bottom) (1×10⁶ cells / wells). 5 (1 cell / well), then add NCU-106 antibody (two concentration gradients of 1 and 1000 ng / mL), pre-incubate at 37°C for 15 minutes, and then add ε-NAD to a final concentration of 50 μM for enzymatic reaction.

[0065] All experimental groups were performed on the same 96-well plate, with a reaction volume of 200 μL / well. Fluorescence signal detection was performed immediately after the addition of ε-NAD. Using a SpectraMax i3x instrument at a constant temperature of 37°C, fluorescence detection was performed in 300 nm excitation and 410 nm emission mode, with fluorescence signals read every 5 minutes for 30 minutes. CD38 / NADase enzyme activity levels were positively correlated with fluorescence values.

[0066] Experimental results (see) Figure 3 The results showed that in two different cell lines, both the wild-type antibody Isatuximab and the anti-CD38 antibody mutant NCU-106 were able to inhibit CD38 / NADase activity in a dose-dependent manner, and the inhibitory activity of the anti-CD38 antibody mutant NCU-106 was significantly stronger than that of the wild-type antibody Isatuximab.

[0067] Example 6: Detection of NCU-106 Affinity and Specificity

[0068] In this experiment, total protein (without high-temperature denaturation) from the human non-small cell lung cancer cell line A549 and the human multiple myeloma cell line RPMI-8226 was isolated using SDS-PAGE. The isolated proteins were then electroporated onto a PVDF membrane and incubated for 4 hours at room temperature with the wild-type antibody Isatuximab and the anti-CD38 antibody mutant NCU-106, respectively. After incubation, the membranes were thoroughly washed to remove unbound antibodies.

[0069] Since both antibodies have a His×6 tag at their C-terminus, the His antibody can be used for detection. Next, the His antibody was added and incubated at room temperature for 4 hours. After rinsing again, the secondary antibody was added, and the signal was displayed using ECL.

[0070] Experimental results (see) Figure 4 The results showed that at the molecular weight position of the CD38 protein, clear bands appeared on the PVDF membranes incubated with both Isatuximab and NCU-106, with no obvious impurities. This indicates that the anti-CD38 antibody mutant NCU-106 retains equivalent affinity and specificity compared to the wild-type antibody Isatuximab.

[0071] Example 7: Detection of the immune effect of the anti-CD38 antibody mutant NCU-106

[0072] This experiment assessed the immune response of the anti-CD38 antibody mutant NCU-106 using antibody-dependent cytotoxicity (ADCC) assay. The ADCC mechanism involves the activation of effector cells by receptors on the surface of immune cells (such as NK cells) binding to the Fc region of the antibody, which in turn induces apoptosis in target cells by secreting substances such as perforin and granzymes.

[0073] Effector cells (E) were human NK cells derived from human peripheral blood lymphocytes. Target cells (T) included RPMI-8226 (human multiple myeloma peripheral blood B lymphocytes) and Raji (human Burkitt's lymphoma cells). NK cells were isolated from whole blood of healthy individuals undergoing physical examinations at the Department of Physical Examination, First Affiliated Hospital of Nanchang University. All sample acquisition was approved and authorized by the Ethics Committee of the First Affiliated Hospital of Nanchang University. Monocytes (PBMCs) were isolated from peripheral blood using lymphocyte separation medium, with samples from up to 10 donors mixed to avoid individual variability. Cells were resuspended in culture medium (containing inactivated serum), and cell viability and count were assessed. ADCC assays were only performed when the viable cell percentage of isolated PBMCs was ≥70%. Fetal bovine serum inactivation was performed by incubation at 56°C for 30 minutes, with several shakes during incubation.

[0074] Logarithmic growth phase RPMI-8226 and Raji cells were pre-incubated with Calcein-AM dye. After the dye entered the live cells, it was cleaved by enzymes, eliciting green fluorescence. Next, after cell counting, the cells were uniformly seeded, and then different concentrations of wild-type antibody Isatuximab and anti-CD38 antibody mutant NCU-106 (0, 1, and 10 μg / mL) were added, followed by pre-incubation at 37°C for 30 minutes. Then, PBMCs (E:T = 20:1) were added, and co-culture continued at 37°C for 5 hours. After culture, the cells were centrifuged, the supernatant was collected, and fluorescence detection was performed using a SpectraMax i3x instrument with an excitation wavelength of 300 nm and an emission wavelength of 410 nm. If cell contents were released, green fluorescence could be detected in the supernatant. Changes in fluorescence intensity reflected the intensity of immunotoxicity.

[0075] The experimental group was set up as follows:

[0076] 1. Culture medium control group: contains only complete culture medium, without cell addition.

[0077] 2. Vehicle group: Blank culture medium was used instead of PBMC cells.

[0078] 3. Isa group: Target cells were incubated with different concentrations of wild-type antibody Isatuximab (0, 1 and 10 μg / mL) at 37°C for 30 minutes, and then PBMCs were added.

[0079] 4. NCU-106 group: After incubating target cells with different concentrations of anti-CD38 antibody mutant NCU-106 (0, 1 and 10 μg / mL) at 37°C for 30 minutes, PBMCs were added.

[0080] 5. Positive control group: Target cells were disrupted by an ultrasonic disruptor, and the supernatant was collected after high-speed centrifugation for direct fluorescence detection.

[0081] Experimental results (see) Figure 5 The results showed that the fluorescence value increased in a dose-dependent manner with increasing dose of the wild-type antibody Isatuximab, suggesting increased release of target cell contents and enhanced immunotoxicity. However, the fluorescence values ​​of the pretreated groups with different doses of the anti-CD38 antibody mutant NCU-106 were comparable to those of the Vehicle group, indicating that they did not have significant immunotoxic effects.

[0082] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An anti-CD38 antibody mutant, characterized in that, The amino acid sequence of the anti-CD38 antibody mutant is based on the wild-type antibody Isatuximab, with the heavy chain of the mutant undergoing mutations of Y27H, G99A, N105D, L237A, L238E, and P332G. The amino acid sequence of the heavy chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.4, and the amino acid sequence of the light chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.

5.

2. The anti-CD38 antibody mutant of claim 1, wherein The gene sequence encoding the heavy chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.8, and the gene sequence encoding the light chain of the anti-CD38 antibody mutant is shown in SEQ ID NO.

9.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the amino acid sequence of the anti-CD38 antibody mutant according to any one of claims 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 3, and the recombinant expression vector is used to express the anti-CD38 antibody mutant.

5. A host cell, characterized in that, The host cell contains the recombinant expression vector as described in claim 4.

6. A method for blocking NAD + The consumed blocking agent is characterized by, The blocking agent comprises an anti-CD38 antibody mutant of claim 1; the blocking agent inhibits the enzymatic catalytic activity of CD38 by specifically binding to CD38, thereby blocking the depletion of NAD + .

7. The NAD blocking method according to claim 6 + The consumed blocking agent is characterized by, It also includes buffer solutions.

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