Targets and Antibody Formats for Immunotherapy of Neuroblastoma – summary

Introduction
 Chemotherapy isn’t a be all solution for metastatic tumours. Immunotherapy,
alongside chemotherapy, has proven to eradicate such tumours, sometimes
completely.
 Moreover, rigorous chemotherapy can impact adaptive and innate immunity.
 Low tumour mutation burden paired with lesser expression of HLA receptors lead to
an impaired T-cell immune response, as seen in neuroblastoma studies.
 Approved immunotherapeutic approaches:
o 33 US FDA approved antibodies
o 2 vaccines (talimogene laherparepvec and sipuleucel-T)
o 2 cell therapies (axicabtagene ciloleucel and tisagenlecleucel)
 High-risk neuroblastoma cases are treated with chemotherapeutics, surgical
intervention, radiation exposure, stem cell transplantation, and immunotherapy
(monoclonal antibodies).
 A combination of radiation and immunotherapy (radioimmunotherapy or RIT) using
iodine-131-8H9 has led to a better prognosis in patients with relapsed neuroblastoma
in CNS.
 Similar results were obtained in patients treated with vaccines anti-GD2 and anti-
GD3.
 Neuroblastoma: immunological properties
o Low risk neuroblastoma: display gain in chromosomes. HLA I antigen is
expressed, which helps in T-cell recognition. OMA Syndrome can also be
seen
o Metastatic neuroblastoma: embedded in TME, amongst macrophages that
specifically act against T cells. Downregulation of HLS I antigen is seen in
this case. Immunosuppressive proteins and sugars are produced. Lymphocytes
undergo programmed cell death.

Methodology
 Target molecules against neuroblastoma:
o Disialogangioside GD2 – leads to an increase in proliferation rate in neuronal
cells
o B7-H3 or CD276 – overexpressed in some tumour cells
o ALK – lymphoma kinase, expressed exclusively in certain cancer cells,
including neuroblastoma
 Immunotherapy against neuroblastoma through specialised antibodies
 Monoclonal IgG antibodies
o Produced using hybridoma technology
 o Anti-GD2 monoclonal antibodies – promotes cell death by cell-mediated
cytotoxicity produced by antibodies (through macrophages and NK cells) and
complement system
 Examples: chimerized antibody produced in myeloma cells of mouse
origin CH14.18 (dinutuximab-beta) or CHO, and 3F8 (naxitamab)
from mouse origin.
 3F8 is known to push the cells to complement system-based
cytotoxicity, while CH14.18 promotes antibody mediated cytotoxicity.
However, both show different toxicity profiles.
 Patients (high-risk neuroblastoma) given 3F8 with colony stimulating
factor for granulocytes-macrophages, had a better survival rate (>65%)
 Similar fates were observed in patients treated with interleukin-2,
retinoic acids, and the same colony stimulating factor
 Other studies on CH14.18 and 3F8 have shown that complement
activation could be crucial for treatment of neuroblastoma
 Anti GD2 antibodies are less active against neuropathic pain, soft-
tissue cancer cells, and other side effects of this immunotherapeutic
approach
 Mutation was induced in CH14.18 antibody at K322A – an increase in
antibody mediated cytotoxicity was observed
o The activity of the monoclonal antibodies was increased using conjugates
(prodrugs, cytokines, and radionuclides)
 Prodrugs are activated in the stromal space of tumours or inside the
targeted cells. Two types: Agents that damage the DNA
(calicheamicin, anthracyclines, pyrrolobenzodiazepines, duocarmycin)
and inhibitors of microtubules (auristatins, maytansines).
 Examples: lorvotuzumab, lorvotuzumab mertansine, m906
combined with pyrrolobenzodiazepines, anti-GD2 antibodies
carrying sepantronium bromide
 Radionuclides alongside antibodies emit electrons or alpha/beta
particles that push the targeted cells towards cell death, hence can also
result in toxicity
 Examples: B7-H3 or GD2 tagged with I radionuclide, 3F8
tagged with I, I-labelled omburtamab, actinium-225 tagged 3F8
that emits alpha particles
 Cytokines given with antibodies lead to an increased immune response
against cancer cells
 Examples: Colony stimulating factorIL-2, IL-12, IL-13, or IL-15
tagged antibodies (either CH14.18 or 3F8)
 Bi-specific antibodies or BsAbs against neuroblastoma
o Are simultaneously specific to a tumour cell specific epitope and either NK
cell or T-cell receptor proteins (CD16 and CD3, respectively)
o Structurally there are two classifications: IgG like and non-IgG like BsAbs
o IgG like BsAbs (>150 kDa) are bigger than non-IgG like BsAbs (<65 kDa),
this reduces the half life of non-IgG like antibodies.
 o IgG like BsAbs show better structural symmetry, easy production, stable drug
delivery, and overall good in-vivo distribution
o Examples: humanized 3F8-BsAb, radiolabelled 3F8-C825 with anti-DOTA,
other emitting radionuclides can also be conjugated with BsAb
Results
 Limitations seen so far in GD2 immunotherapy
o Although the anti-GD2 monoclonal antibodies have proven to be quite
efficient in treating neuroblastoma, protein engineering can still bring crucial
changes in the existing strategies
o T-BsAbs have improved anti-GD2 activity, same goes for BiTE producing
virus combined with T-cell therapy
o Combinations of antibodies and prodrugs, radionuclides or other such agents
has shown an impressive increase in survival rate in preclinical studies
 Chemotherapy and radiotherapy negatively impact immune system
o Due to prolonged exposure to the classical treatment options such as chemo-
and radiotherapy, immune cells are inadequate in mounting an appropriate
response against the tumour cells
o But this can be circumvented by combining cytokines with antibodies (IL-15
has pleiotropic effects on T cells and NK cells)
o Cytokine conjugated antibodies can be injected directly into the tumour cells
to increase the overall efficacy
 Immunosuppressive TME or tumour micro-environment can be combated using anti-
CD105 antibodies, vorinostat conjugated antibodies
 NK cell receptors and other molecules can be specifically targeted to improve the
immune response
 Immunotherapy can of course be combined with radiation or chemotherapy. In such
case antibodies can be tagged and directly injected at the site to reduce possible
cytotoxicity (on-target, off-tumour)
 This review focuses on a few antibody-based therapies (like anti-GD2, anti-NCAM,
CD56, ALK, B7-H3, glypicans and polysialic acid
 Studies on antigens present inside the cancer cells that can be presented through HLA
have just started (such as oncoproteins like MYCN, transcription factor, telomerase
and so on)
Conclusion
 Immunotherapy against neuroblastoma cells has opened a new avenue of treatment of
cancer patients.
 It is also a great alternative or complementary to chemotherapy, with less side effects
from toxicity
 Our understanding about the exact mechanism of the therapeutics however is still
lacking which asks for more in-depth studies to be designed and conducted
 New and novel antibodies and their conjugates can also be designed, based on the
available data
 Antibody mediated treatments can especially prove to be an indispensable option in
metastatic neuroblastoma in children