Patent Application
20230364257
The sequence listing provided in the file entitled C6850-018_SQL_REV.xml, which is an Extensible Markup Language (XML) file that was created on Jun. 20, 2023, and which comprises 3,499 bytes, is hereby incorporated by reference in its entirety.
Technical Field: The present invention pertains to the field of pharmaceutical engineering and specifically relates to an antibody conjugate and a method for enhancing antibody molecular immune effector functions.
Malignant tumors continue to pose a major threat to human health, with both the incidence and mortality rates on the rise. Monoclonal antibodies are among the most effective biopharmaceuticals used in anti-cancer therapy. They exert therapeutic effects by targeting tumor-specific antigens or related antigens, primarily through mechanisms such as inducing or blocking cell signaling, activating immune effector functions, and serving as carriers for specific drug delivery to target cells. Among these mechanisms, immune effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), play a crucial role and represent the fundamental functionality of most anti-tumor antibody drugs. Therefore, enhancing the ADCC and CDC activities of antibodies through antibody engineering has become a hot topic in antibody biopharmaceutical research and development.
Currently, antibody engineering techniques are predominantly used to enhance antibody ADCC and CDC activities, leading to the development of next-generation monoclonal antibodies with improved immune activities. However, these techniques are complex and require extensive screening to obtain antibodies with satisfactory immunoreactivity, resulting in significant workloads. Moreover, modified antibodies exhibit a certain degree of immunogenicity, leading to immune-related side effects.
There are naturally occurring antibodies in the human body that are specific to certain haptens. Examples of such antibodies with relatively high abundance include antibodies against Rha (rhamnose), DNP (2,4-dinitrophenol), and αGal (alpha-galactose). Modifying commercial monoclonal antibodies with these haptens holds promise for enhancing the immune effector functions of the original antibodies. For instance, it has been reported that conjugating αGal derivatives to anti-CD20 antibodies can yield CD20 antibodies with enhanced CDC levels. However, this approach is hindered by complex modification methods that can affect antibody affinity, product stability, and yield. Additionally, it fails to assess the ADCC levels of the resulting antibodies and optimize the length of critical factors such as the linker, ultimately limiting the improvement of immune effector function levels.
Therefore, there is an urgent need for a novel method to enhance antibody immune effector functions, addressing or mitigating the aforementioned issues.
The purpose of the present invention is to overcome the limitations of existing technologies. It provides an antibody conjugate and a method for enhancing immune effector functions of antibody, based on a strategy involving the recruitment of naturally occurring hapten-specific antibodies. Furthermore, by optimizing the linker, the invention offers an optimized selection of hapten derivative molecules that result in antibody conjugate with significantly improved immune effector functions compared to previously reported antibodies.
To achieve the aforementioned objectives, the present invention employs the following technical solutions:
In some aspects, the present disclosure provides an antibody conjugate, comprising an antibody modified by a hapten derivative molecule. The hapten derivative molecule consists of a hapten, a linker, and a coupling domain.
In some aspects, the present disclosure provides the antibody conjugate, wherein the hapten is a small molecule capable of recruiting specific natural antibodies in the human body. Examples of such haptens include, but are not limited to, Rha, DNP, and αGal.
In some aspects, the present disclosure provides the antibody conjugate, wherein the linker comprises, but is not limited to, a polyethylene glycol linker, alkyl chain or aliphatic chain linker or spacer ((CH2)n), and a peptide linker or spacer. In some aspects, to facilitate the synthesis of homogeneous products and minimize undesired toxic side effects, the preferred linker arm comprises, but is not limited to, a PEG with low molecule weight (less then 1000), a short alkyl chain or aliphatic chain linker (molecular weight less than 100) and a peptide linker, and further, in some embodiments, low polyethylene glycol PEGS is preferred.
In some aspects, the coupling domain of the invention is used to modify hapten derivatives on antibodies.
In some embodiments, in order to facilitate the synthesis of uniform products and reduce the impact of hapten derivatives on the original affinity of antibodies, the coupling site is preferably the eight thiol groups formed by the reduction of four disulfide bonds on the antibodies; In some embodiments, the coupling domain is preferably SMCC, or other molecules containing maleimides
or other molecules containing vinyl sulfones
or other molecules containing acrylates or acrylamides
or other molecules containing methacrylates,
R and R′ is one or more substituents.
In some aspects, the present disclosure provides the antibody conjugate, wherein the antibody is a monoclonal antibody, including but not limited to anti-CD20 antibody, anti-CD19 antibody, anti-CD30 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, anti-HER2 antibody, anti-HER3 antibody, anti-PSMA antibody, anti-VEGFR antibody, anti-PD-L1 antibody, anti-cMET antibody, anti-TGF-β antibody, anti-MUC1 antibody, and anti-Trop-2 antibody.
In some embodiments, the present disclosure provides the antibody conjugate, wherein the heavy chain of antibody is represented by SEQ ID NO.1, and the light chain of antibody is represented by SEQ ID NO.2.
In some embodiments, the present disclosure provides the antibody conjugate, wherein the hapten derivative includes one of the following structures:
In some embodiments, the present disclosure provides the antibody conjugate, wherein the synthetic pathway of the hapten derivative is:
wherein n is a positive integer.
Another objective of the present invention is to provide a method for systhesizing the antibody conjugate with enhanced immune effector functions of antibodies by modifying antibodies with hapten derivative molecules, thereby utilizing the recruitment ability of naturally occurring antibodies specific to the hapten molecules to enhance immune activities such as ADCC and CDC. Furthermore, the invention aims to optimize the linker of the hapten derivative molecules to obtain antibody conjugates with significantly improved immune effector functions.
The hapten derivative molecules, antibodies, modification sites, and methods for enhancing the molecular immune effector functions of antibodies are as previously described.
In an embodiment of the present invention, a method for preparing the antibody conjugate by modifying the hapten derivative onto the antibody, comprising the following steps:
1) Desalting the antibody in a sodium borate reaction.
2) Adding a thiol-containing reducing agent TCEP and reacting under dark and shaking conditions.
3) After the reaction is completed, adding a 10-fold excess of the hapten derivative and quenching the reaction with cysteine.
4) Ultrafiltration to remove excess small molecules and obtain the corresponding antibody conjugate.
In a specific embodiment of the present invention, the selected antibody is rituximab, a monoclonal antibody against CD20 (KEGG Accession Number: DB00073). The heavy chain is represented by SEQ ID NO.1, and the light chain is represented by SEQ ID NO.2. CD20 is highly expressed on the surface of over 95% of B-cell lymphoma cells, making it an important target for monoclonal antibody-based therapies. The optimized hapten derivative molecule is SMCC-PEG3-Rha
where SMCC represents the coupling domain, PEG3 represents a polyethylene glycol chain, and Rha represents the hapten. The coupling site is the eight thiol groups formed after reducing the four disulfide bonds on rituximab antibody.
The Advantages of the Present Invention Compared to Prior Art:
(1) The antibody conjugates described in the present invention exhibit both unimpaired antigen affinity levels compared to reported antibodies and significantly enhanced ADCC and CDC activities. This allows for lower dosages during treatment, ultimately reducing or avoiding the occurrence of certain toxic side effects.
(2) The optimized hapten derivative molecules provided in the present invention lead to antibody conjugates with the most prominent immune effector functions. These conjugates demonstrate significantly higher CDC killing effects compared to wild-type antibodies, using only 4% of the dosage of the wild-type antibody.
(3) The strategy employed in the present invention to enhance antibody immune effector functions is not only simple to implement and utilizes mild modification conditions but also avoids potential toxicity and immunogenicity by harnessing the body's own naturally occurring antibodies.
The present invention discloses a class of antibody-hapten conjugates, and a method to magnify the immunity effector functions of antibody. Antibody-hapten conjugates are antibody molecules modified with hapten derivates, and hapten derivates are synthesized small molecules capable of binding to the existing antibodies in human serum. The present invention provides a simple and east method to generate antibody-hapten conjugates. Theses conjugates retain excellent binding specificity and binding affinity, and are able to recruit the existing antibodies onto the cancer cell surface and further form an immune complex that is able to provide multivalent Fc domains to interact with immune cells or complement protein C1q, leading to magnified ADCC and CDC simultaneously. Moreover, the present invention also provides conjugates with optimal ADCC and CDC activities according to the structure—activity relationship.
The following embodiments are used to describe the present presentation. However, they are not intended to limit the scope of the present invention. Any simple improvement under the conception of the present invention is within the scope of protection of the present invention. All experimental methods in the following embodiments are conducted according to the procedures provide by companies unless otherwise stated. All the materials and cell lines in the following embodiments are purchased commercially unless otherwise stated.
In the present embodiments, SMCC, PEG and Rha are selected as the coupling domain, the linker and the hapten, respectively. According to the synthesis methods of the antibody-hapten conjugates in the present invention, the hapten derivatives SMCC-PEGn-Rha (n=1, 3, 6) are firstly synthesized. Then, hapten derivates are coupled to the antibody molecules to generate the corresponding antibody conjugates. Notably, the structure—activity relationship between the length of PEG linker and the biological activities of conjugates was evaluated.
The details, including the coupling domain, the linker and the hapten in the following embodiments, are used to describe the present presentation. However, they are not intended to limit the scope of the present invention.
Containing Different PEG Linkers
(1) The synthesis procedure of hapten derivate SMCC-PEG1-Rha is as follows:
The observed 1H-NMR, 13C NMR and HRMS of SMCC-PEG1-Rha are all in line with the calculated theoretical date. SMCC-PEG1-Rha: 1H NMR (400 MHz, Methanol-d4) δ 6.80 (s, 2H), 4.66 (d, J=1.7 Hz, 1H), 3.79 (dd, J=3.5, 1.7 Hz, 1H), 3.69 (dt, J=10.0, 5.7 Hz, 1H), 3.63 (dd, J=9.5, 3.5 Hz, 1H), 3.58-3.51 (m, 1H), 3.45 (ddd, J=10.3, 6.1, 4.7 Hz, 1H), 3.40-3.31 (m, 6H), 2.14 (tt, J=12.2, 3.5 Hz, 1H), 1.82 (dd, J=13.5, 3.4 Hz, 2H), 1.76-1.69 (m, 2H), 1.42 (qd, J=13.1, 3.3 Hz, 2H), 1.24 (d, J=6.2 Hz, 3H), 1.00 (qd, J=13.1, 3.5 Hz, 2H). 13C NMR (101 MHz, Methanol-d4) δ 177.68, 171.37, 133.87, 100.27, 72.53, 70.94, 70.75, 68.48, 65.55, 44.70, 43.12, 38.74, 36.49, 29.58, 28.66, 16.60. HRMS (ESI, positive) calculated for C20H30N2O8Na [M+Na]+: 449.1900 Found: 449.1899.
(2) The synthesis procedure of hapten derivate SMCC-PEG3-Rha is as follows:
The observed 1H-NMR, 13C NMR and HRMS of SMCC-PEG3-Rha are all in line with the calculated theoretical date. SMCC-PEG3-Rha: 1H NMR (400 MHz, Methanol-d4) δ 6.71 (s, 2H), 4.62 (d, J=1.7 Hz, 1H), 3.73-3.65 (m, 2H), 3.57-3.49 (m, 8H), 3.43 (t, J=5.5 Hz, 2H), 3.30-3.19 (m, 6H), 2.05 (tt, J=12.1, 3.4 Hz, 1H), 1.72 (dd, J=13.5, 3.5 Hz, 2H), 1.66-1.60 (m, 2H), 1.38-1.20 (m, 3H), 1.16 (d, J=6.2 Hz, 3H), 0.91 (qd, J=12.9, 3.5 Hz, 2H). 13C NMR (101 MHz, Methanol-d4) δ 133.88, 100.41, 72.59, 70.99, 70.82, 70.26, 70.05, 69.27, 68.41, 66.30, 48.27, 48.05, 47.84, 47.63, 47.41, 47.20, 46.99, 44.67, 43.14, 38.86, 29.57, 28.63. HRMS (ESI, positive) calculated for C24H38N2O10Na [M+Na]+: 537.2424 Found: 537.2415.
(3) The synthesis procedure of hapten derivate SMCC-PEG6-Rha is as follows:
The observed 1H-NMR, 13C NMR and HRMS of SMCC-PEG6-Rha are all in line with the calculated theoretical date. SMCC-PEG6-Rha: 1H NMR (400 MHz, Methanol-d4) δ 6.81 (s, 2H), 4.71 (d, J=1.7 Hz, 1H), 3.82-3.75 (m, 2H), 3.68-3.57 (m, 22H), 3.52 (t, J=5.5 Hz, 2H), 3.40-3.31 (m, 5H), 2.15 (tt, J=12.2, 3.6 Hz, 1H), 1.82 (dd, J=13.7, 3.5 Hz, 2H), 1.76-1.69 (m, 2H), 1.42 (qd, J=13.1, 3.3 Hz, 2H), 1.25 (d, J=6.2 Hz, 3H), 1.01 (qd, J=12.9, 3.6 Hz, 2H). 13C NMR (101 MHz, Methanol-d4) δ 171.39, 133.89, 100.43, 72.61, 70.98, 70.81, 70.28, 70.22, 70.20, 70.18, 70.16, 70.13, 70.02, 69.88, 69.19, 68.41, 66.40, 44.67, 43.14, 38.86, 36.51, 29.60, 28.63, 16.67. HRMS (ESI, positive) calculated for C30H51N2O10 [M+H]+: 647.3391 Found: 647.3389.
The procedures for preparing rituximab-Rha conjugates are as follows:
1) Rituximab was exchanged into 25 mM of sodium borate buffer.
2) Tris(2-carboxyethyl)phosphine (TCEP) was added to break the four pairs of disulfide bonds in dark, the reaction progress was monitored by Ellman's analysis.
3) The reduced Rituximab was conjugated with 10 equiv of SMCC-PEG1-Rha, SMCC-PEG3-Rha, or SMCC-PEG6-Rha, then the reaction was quenched using cystine.
4) Excess hapten derivates were removed by ultrafiltration to give the final rituximab-Rha conjugates αCD20-PEG1-Rha, αCD20-PEG3-Rha and αCD20-PEG6-Rha.
The conjugates were further characterized by LC-MS analysis. As shown in
The procedure of flow cytometry is as follows:
1) Raji cells (CD20 positive) and K562 cells (CD20 negative) were collected and resuspended to 4×105/mL. Then, 100 μL of these cells was added into tubes containing 50 nM of antibody samples. The incubation was conducted on ice for 30 min.
2) Cells were incubated with FITC-conjugated goat anti-human IgG antibodies (for binding affinity assay), or successively treated with 1% anti-Rha rabbit serum and FITC-conjugated goat anti-rabbit IgG antibodies (for antibody-recruiting ability assay).
3) Cells were finally detected using the Accuri C6 flow cytometer.
As shown in
The results of antibody-recruiting ability were presented in
The ADCC and CDC mediated by rituximab-Rha conjugates in the absence or presence of anti-Rha antibodies were all evaluated.
ADCC assay: 1) Raji cells were collected and resuspended to 4×105/mL. Then, 50 μL of these cells was added to each well in 96-well plates. 2) Immediately supplemented with 50 μL different concentrations of antibody samples (or PBS as the negative control), 50 μL of 4% anti-Rha antibodies and 50 μL of freshly isolated human peripheral blood mononuclear cells (PBMCs), the incubation was performed at 37° C. for 4 h. 3) The cytotoxicity was determined using an LDH kit.
ADCC results were displayed in
CDC results were displayed in
According to the above embodiments, rituximab-Rha conjugate αCD20-PEG3-Rha existed the highest ADCC and CDC activities, in another words, has made a tremendous improvement for ADCC and CDC. By contrast, conjugate αCD20-PEG1-Rha containing a shorter PEG linker and αCD20-PEG6-Rha containing a longer PEG linker both showed lower ADCC and CDC activities. This difference may be explained by the steric hindrance which limit the interaction of αCD20-PEG1-Rha and anti-Rha antibodies, resulting in the lower antibody-recruiting level. On the other hand, this difference may be also explained by the unexpected reduced affinity of αCD20-PEG6-Rha against anti-Rha antibodies, resulting in restricted ADCC and CDC activities.
The above embodiments are used to describe the present presentation. However, they are not intended to limit the scope of the present invention. Any simple improvement under the conception of the present invention is within the scope of protection of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202011516850.9 | Dec 2020 | CN | national |
The present application is a Continuation of International Application Number PCT/CN2021/137352 filed Dec. 13, 2021, and claims priority to Chinese Application Number 202011516850.9 filed Dec. 21, 2020.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/137352 | Dec 2021 | US |
| Child | 18337631 | US |
