Carefully crafted, hit the weakness!

It was a golden age everywhere.

In the 1970s, the research and development of antibody drugs opened the Nuggets era.

Stuart Schlossman, a famous immunologist at Harvard Medical School, became the fastest sharpshooter in this wild west game [1].

Standing at that historical stall, the grand blueprint of antibody drugs has a vague outline.

First of all, the key scientific problems of antibody structure and biological characteristics have been solved one by one; secondly, in 1975, Georges Köhler and César Milstein published the revolutionary hybridoma technology in the top journal Nature [2], which will bring large-scale The dream of antibody preparation shines into reality.

Georges Köhler and César Milstein won the 1984 Nobel Prize in Physiology or Medicine for hybridoma technology.
At this time, Professor Schlossman was obsessed with studying the biological characteristics of T cells.

In 1979, Schlossman and his collaborators identified three monoclonal antibodies against T cell antigens, one of which was called OKT3.

Soon, researchers discovered that OKT3 can be used to deplete T cells.

This also means that OKT3 may become a powerful player in the treatment of immune rejection.

The soldiers are very fast.

In 1981, OKT3, as an immunosuppressant, opened a clinical trial for the treatment of transplant rejection.

Five years later, OKT3 (later renamed muromonab-CD3) was approved by the FDA in one fell swoop, becoming the world's first therapeutic monoclonal antibody approved for marketing.

40 years after the first monoclonal antibody was born, in April 2021, the PD-1 inhibitor dostarlimab was approved for marketing, becoming the 100th monoclonal antibody approved by the FDA for marketing [3].

FDA's monoclonal antibody drug approval history data at a glance 40 years, 100 new drugs, monoclonal antibody technology has completely changed the tide of drug development and disease treatment.

However, 40 years is just a comma.

The combination of fusion, antibody coupling technology for grafting cytotoxic drugs, double-targeted bispecific antibodies, and antibody engineering to make classic monoclonal antibody drugs better and stronger, are shaping the broader future of antibody drugs.

This is still a golden age everywhere.

The weakness of classic antibodies Among the 100 antibody drugs approved by the FDA, 42% of the antibodies target ten popular targets, and HER2 is one of the most dazzling targets.

Trastuzumab, which targets HER2, is also a model of antibody development in the Nuggets era.

In 1979, the team of Professor Robert A.
Weinberg first discovered the HER2 gene [4].

Eight years later, Dr.
Dennis Slamon of the University of California, Los Angeles, together with Dr.
Bill McGuire of Texas State University, San Antonio, and several Genentech scientists discovered that about 20%-30% of breast cancers have HER2 gene amplification.
Increase or overexpression [5], HER2 gene has become a powerful target for the treatment of breast cancer.

Ten years later, the FDA approved trastuzumab for the treatment of HER2-positive metastatic breast cancer.

The birth of trastuzumab, the top ten target among 100 monoclonal antibody drugs, has completely changed the fate of patients with HER2-positive advanced breast cancer.

But there is always imperfection in perfection.

Subsequent studies have found that not all patients receiving trastuzumab treatment can obtain good treatment results.

Some clinical trials also suggest that specific genotypes can predict the therapeutic effect of trastuzumab.

We know that therapeutic monoclonal antibodies can induce tumor cell death through direct or indirect mechanisms.

Direct mechanisms include blocking growth factor receptor signal transduction, direct transmembrane signal transduction, etc.
; indirect mechanisms need to cooperate with other members of the host immune system, including complement-mediated cytotoxicity (CDC), antibody-dependent cells Mediated cell phagocytosis (ADCP), and antibody-dependent cell-mediated cytotoxicity (ADCC) [6].

From the perspective of monoclonal antibody structure, the direct mechanism of action mainly depends on the antibody-binding fragment Fab, which can specifically recognize the relevant antigen on the surface of tumor cells, thereby regulating the signal pathway related to the antigen.

The indirect mechanism mainly depends on the crystallizable region Fc.

The structure of monoclonal antibody drugs.
For trastuzumab, the direct mechanism includes receptor internalization, receptor shedding, direct anti-proliferative activity, etc.
; while the indirect mechanism is mainly ADCC [7].

For trastuzumab belonging to IgG1 antibody, the Fc receptor family-FcγR plays a key role in the ADCC mechanism.

In ADCC, the Fab fragment of the antibody binds to the target on the tumor cell, and the Fc fragment is recognized by the FcγR on the effector cell.

The interaction between Fc and FcγR activates effector cells, prompting them to release cytotoxic particles, which leads to tumor cell lysis.

The FcγRs family has three brothers, namely FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) [8,9].

Among them, the most closely related to ADCC are the activated receptor CD16A (a subtype of CD16) and the inhibitory receptor CD32B (a subtype of CD32).

In other words, the higher the affinity of the antibody to CD16A, the stronger the ADCC effect; the stronger the affinity to CD32B, the weaker the ADCC effect.

In the FcγRs family, the most closely related to ADCC is the activating receptor CD16A (a subtype of CD16) and the inhibitory receptor CD32B (a subtype of CD32).
Follow-up studies have found that the 158th amino acid of CD16A contains valine ( V) or phenylalanine (F) genetic polymorphism will greatly affect its affinity for IgG1.

Studies have shown that the CD16A-V158 allele has a higher IgG1 binding affinity than the CD16A-F158 allele.

In the in vitro ADCC analysis, the CD16A-V158 allele also showed stronger ADCC activity [10].

The difference in antibody affinity of different genotypes eventually translates into differences in therapeutic effects.
Studies have shown that patients who are homozygous for the CD16A-V158 allele receive trastuzumab treatment with better progression-free survival than patients with other genotypes [11, 12].

So, how many people are lucky enough to be homozygous for CD16A-V158? Studies have shown that CD16A-V158 homozygotes account for 10% to 20% of the world’s population [13,14].

In other words, for the vast majority of patients receiving trastuzumab, the ADCC effect induced by antibodies still has a lot of room for improvement.

The question before scientists is, can we improve the ADCC effect of the antibody by modifying the Fc segment of the antibody? The evolution of the artistic life of antibody micro-sculpting has given us humans an extremely delicate and magnificent immune system.

Every day, our human body produces about 1 billion B cells, and each B cell produces a unique antibody.

All antibodies belong to five types of immunoglobulins, which are IgG, IgA, IgM, IgD, and IgE.
Among the immunoglobulins in human serum, 60% are IgG.

Almost all monoclonal antibody drugs approved by the FDA belong to IgG[15].

Schematic diagram of the indirect mechanism of monoclonal antibody drugs IgG immunoglobulins are of a unique Y type, the upper Fab segment is responsible for binding to the antigen, and the lower Fc segment is responsible for binding to other immune effector cells.

In recent years, through engineering the Fc segment to achieve a leap in the therapeutic effect of antibodies, it has become a strong force in the research and development of antibody drugs.

The engineering of various different pathways can achieve a series of antibody performance improvements such as half-life optimization and ADCC effect improvement through precise fine-tuning of several amino acids in the Fc segment of antibodies or their modification methods, which can be regarded as an art of antibody micro-sculpting.
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Taking glycosylation as an example, we know that human IgG has two biantennary glycans located at Asn297.
The core is composed of N-acetylglucosamine and mannose, and the periphery is composed of fucose, galactose, etc.

In the process of commercial production of antibodies, glycosylation is highly heterogeneous.
The presence and composition of glycosyl groups will affect the conformation of Fc to a large extent, thereby further affecting the binding ability of Fc and FcγR.

Studies have shown that removing fucose can significantly enhance the affinity of antibodies to CD16A, thereby enhancing ADCC activity.

Therefore, a large number of glycosylation modifications based on the removal of fucose are being tested in different antibody drugs.

In addition to glycosylation modification, scientists are also using different methods to find amino acid sites that can modify the performance of the Fc segment of antibodies.

The traditional alanine scanning library technology uses the characteristics of alanine that is small in size and has little effect on the structure of the protein, and the remaining 19 non-alanine residues are replaced with alanine one by one.

Since the replacement of other key amino acids with alanine will cause the weakening or decline of certain functions of the protein, the role of certain amino acid residues in protein function, active site, stability and morphology can be identified.

In addition to alanine scanning technology, scientists also try to use computer algorithm analysis to predict the role of certain key amino acid residues.

In 2007, scientists at MacroGenics used yeast display technology and discovered several amino acid residues that are essential for ADCC effects [16].

By introducing five site mutations such as F243L/R292P/Y300L/L235V/P396L into the Fc segment of the trastuzumab-like antibody, scientists from MacroGenics have developed an engineered antibody Magituximab for trastuzumab Monoclonal antibody (margetuximab).

Further research on the important Fc amino acid residue positions identified by the yeast display technology found that magituximab and trastuzumab have the same HER2 binding ability [17], indicating that the modification of the Fc segment of the antibody has no effect Antigen binding capacity.

Magituximab and trastuzumab have the same HER2 binding ability.
Compared with the wild-type Fc domain, the affinity of magituximab to the two alleles of the activated receptor CD16A Significantly increase, while the affinity with the inhibitory receptor CD32B decreased significantly.

The affinity of magituximab to the two alleles of the activating receptor CD16A increased significantly, while its affinity to the inhibitory receptor CD32B decreased significantly.

The alteration of the antibody's affinity for FcγR after modification also directly affects the ADCC effect induced by the antibody.

Compared with wild-type Fc domain antibodies, magituximab can trigger a stronger ADCC effect.

At the same time, in transgenic mice carrying human CD16A-F158, magituximab also showed higher anti-tumor activity.

So, can the improvement of the ADCC effect of antibody microsculpture be further transformed into real clinical benefits? After all, any structural modification related to the improvement of drug efficacy will be futile if it is not finally reflected in the clinical benefit.

Only with strong clinical benefit data can the pre-clinical hypothesis be finally verified.

In August 2015, Magituximab initiated a head-to-head SOPHIA Phase 3 clinical trial with trastuzumab in 536 patients in 17 countries/regions [18].

536 breast cancer patients who had received more than two anti-HER2 treatments and had disease progression were randomly assigned to receive magituximab + chemotherapy (n = 266) or trastuzumab + chemotherapy (n = 270) .

The results showed that compared with trastuzumab combined with chemotherapy, magituximab combined with chemotherapy significantly reduced the risk of disease progression or death (HR = 0.
76; 95% CI, 0.
59-0.
98; P = 0.
033).
The median progression-free survival periods were 5.
8 months and 4.
9 months, respectively.

Magituximab combined with chemotherapy significantly reduces the risk of disease progression or death.
This is also the first anti-HER2 targeted therapy that has achieved positive results in a head-to-head phase three study with trastuzumab combined with chemotherapy.

On December 16, 2020, the U.
S.
Food and Drug Administration (FDA) officially approved the marketing of magituximab in combination with chemotherapy for the treatment of adult patients with metastatic HER2-positive breast cancer.
These patients have received at least two kinds of antibodies.
HER2 regimen treatment, and at least one regimen is for metastatic disease.

I believe that in the near future, the art of antibody micro-sculpting will also bring more benefits to Chinese patients.

References: 1.
Dustin M L.
Opening the Frontier of the T Cell Surface: Schlossman and Goldstein[J].
The Journal of Immunology, 2013, 190(11): 5343-5345.
2.
KÖHLER, G.
, MILSTEIN, C.
Continuous cultures of fused cells secreting antibody of predefined specificity.
Nature 256, 495–497 (1975) 3.
Mullard A.
FDA approves 100th monoclonal antibody product.
Nat Rev Drug Discov.
2021 May 5.
4.
Bazell R.
Her-2: The making of herceptin, a revolutionary treatment for breast cancer[M].
Random House, 2011.
5.
Slamon DJ, Clark GM, Wong SG, et al.
Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene [J].
science, 1987, 235(4785): 177-182.
6.
Jiang XR, Song A, Bergelson S, Arroll T, Parekh B, May K, Chung S, Strouse R, Mire-Sluis A, Schenerman M.
Advances in the assessment and control of the effector functions of therapeutic antibodies.
Nat Rev Drug Discov.
2011 Feb;10(2):101-11.
7.
Spector NL, Blackwell KL: Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2 -positive breast cancer.
J Clin Oncol 2009, 27:5838-5847.
8.
Siberil, S.
et al.
FcγR: the key to optimize therapeutic antibodies? Crit.
Rev.
Oncol.
Hematol.
62, 26–33 (2007).
9 .
Nimmerjahn, F.
& Ravetch, J.
Fcγ receptors as regulators of immune responses.
Nature Rev.
Immunol.
8, 34–47 (2008).
10.
Lazar, GA et al.
Engineered antibody Fc variants with enhanced effector function.
Proc .
Natl Acad.
Sci.
USA 103, 4005-4010 (2006).
11.
HurvitzSA,BettingDJ,SternHM,etal.
Analysis of Fcγ receptor IIIa and IIa polymorphisms:lack of correlation with outcome in trastuzumab-treated breast cancer patients.
Clin Cancer Res.
2012;18(12): 3478-3486.
12.
NortonN,OlsonRM,PegramM,etal.
Association studies of Fcγ receptor polymorphisms with outcome in HER2+ breast cancer patients treated with trastuzumab in NCCTG (Alliance) Trial N9831.
Cancer Immunol Res.
2014;2(10):962-969.
13.
Sullivan KE, Jawad AF, Piliero LM, Kim N, Luan X, Goldman D, Petri M: Analysis of polymorphisms affecting immune complex handling in systemic lupus erythematosus.
Rheumatology (Oxford) 2003, 42:446-452.
14.
yogoku C, Dijstelbloem HM, Tsuchiya N, Hatta Y, Kato H, Yamaguchi A,Fukazawa T, Jansen MD, Hashimoto H, van De Winkel JG, Kallenberg CG, Tokunaga K: Fcgamma receptor gene polymorphisms in Japanese patients with systemic lupus erythematosus: contribution of FCGR2B to genetic susceptibility.
Arthritis Rheum 2002, 46:1242-1254.
15.
Brezski, RJ & Georgiou, G.
Immunoglobulin isotype knowledge and appli- cation to Fc engineering.
Curr.
Opin.
Immunol.
40, 62–69 (2016).
16.
Stavenhagen JB, Gorlatov S, Tuaillon N, et al.
Fc optimization of therapeutic antibodies enhances their ability to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcγ receptors[J].
Cancer research, 2007, 67(18): 8882 -8890.
17.
Nordstrom JL, Gorlatov S, Zhang W, et al.
Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties[J].
Breast Cancer Research, 2011, 13(6 ): 1-14.
18.
Rugo HS, Im SA, Cardoso F, et al.
Efficacy of margetuximab vs trastuzumab in patients with pretreated ERBB2-positive advanced breast cancer:a phase 3 randomized clinical trial[J].
JAMA oncology, 2021, 7(4): 573-584.
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