Trial Information

Proteome-based Personalized Immunotherapy of Brain Metastases From Lung Cancer

The trial will include 60 cases of refractor brain metastases from lung cancer (BMLC) of
different malignancy after no less than one line of standard chemotherapy and complete
course of radiotherapy.

The participants will enter the experimental (first line therapy) arm and will be subdivided
into 3 subgroups of 20 cases: group I of histologically confirmed lung adenocarcinoma cases
with brain metastases; group II of histologically confirmed small cell lung cancer cases
with brain metastases; and group III of squamous cell lung cancer cases with brain
metastases.

The first line therapy of BMLC involves allogeneic haploidentical hematopoietic stem cells
(HSCs), dendritic vaccine (DV) and cytotoxic lymphocytes (CTLs).

HSCs are used to stimulate individualized adoptive immune response, to affect tumor cells
(TCs) toxically and to regulate tumor stem cells (TSCs) targetedly in order to suppress
their reproductive and proliferative potential. To obtain HSC the donor receives 8
subcutaneous administrations of granulocyte colony-stimulating factor (G-CSF) with 8-10
hours interval for 4 days. The first three days a single dose is 2.5 mcg per 1 kg weight,
the last day the dose is doubled. The stem cells are harvested at day 5. Red blood cells are
withdrawn by centrifuging. The content of cell markers is evaluated by flow cytometry. The
result is assessed after cytoconcentrate enrichment and removal of mature cells and plasma
from it. The preparation is stored in tubes per 4 ml with cryoprotector and 10% poliglucin
solution. Stem cell proportion is no less than 0.5x106 CD34+, and lymphocytes proportion is
no less than 0.5x109 per one administration.

The sample of brain tumor is obtained through stereotaxic/endoscopic/open biopsy from all
patients included into the trial. TCs and TSCs are immunochemically isolated from BMLC
biopsy sample. One part of tumor sample is used for standard histological, cytological and
immunochemical testing, while TCs and TSCs (CD133+) are isolated from the other part.

Dendritic cells are isolated from peripheral blood mononuclear cells and cultured. Tumor
sample provides tumor specific antigens to prepare the dendritic vaccine (DV).

Preparation of CTLs aims to enhance cytotoxic effect on tumor due to great number of
circulating CTLs. CTLs` are isolated from about 100 ml of peripheral blood after 3 DV
administrations, and of them dendritic cells (DCs) are grown. Then, peripheral blood is
repeatedly taken, and lymphocytes are isolated. The CTLs are co-cultured with DCs loaded
with tumor antigens (first line therapy) or recombinant proteins analogous to key
oncospecific proteins (second line therapy) for several times to expand their number
(108-109). Their immunophenotype is detected and CTLs are cryopreserved. The first
stimulation of CTLs with DCs lasts 6-8 days, the second lasts 2-4 days, next 2 days the
lymphocytes are stimulated for the third and fourth time. And then the received lymphocytes
are stimulated by IL-2 for 2 days.

Six months after the first line therapy completion the efficiency is evaluated and the
cohort demonstrating efficiency continues the therapy, while cohort demonstrating no
efficiency will continue the trial with the second line therapy in active comparator arm.

The second line therapy arm uses DV with recombinant proteins analogous to key oncospecific
proteins, autologous CTLs and autologous HSCs with modified proteome.

Autologous HSCs are received from the trial participant as described previously. Cell
preparation of HSC for active comparator arm is obtained of the cytoconcentrate of
autologous mononuclear cells of peripheral blood after mobilization as specified for
experimental arm. Tumor specific antigens for active comparator group are provided by tumor
tissue of the patient.

TCs and TSCs as well as HSCs of the patient undergo complete transcriptome mapping and gene
expression profiling (CTMGEP) and proteome mapping and protein profiling (PMPP). Key (3 or 4
proteins with maximal normalized intensity) oncospecific proteins (OSP) are determined
according to proteome testing of TCs, while proteome profiling of TSCs and use of databases
of protein-protein relations permit detection of intracellular signal transduction pathways
(ISTP) unaffected by carcinogenesis and capable of regulation. Also, receptor membrane
targets to affect these signal pathways (acceptor membrane proteins) are detected, as well
as proteins that are able to activate them (protein ligands). CTMGEP of TSCs confirms
diagnosed functional ISTP. Mathematical modeling of CTMGEP and comparison with Affymetrix
GeneChip Human genome U133A Array data reveals perturbagens able to chemically induce HSCs
and to modify their proteome profile in order to provide secretion of requisite protein
ligands. The database analysis permits understanding of how changes in gene expression
induced by a low-molecular agent or micro RNA corresponds with the changes observed in the
examined profile. If correspondence is significant, it is supposed, that the agent or
similar agents can initiate the effect. If anticorrelation is significant, the agent is
supposed to initiate an opposite effect in gene expression modification. The transcriptome
of HSCs is modified by co-culturing mononuclear cells with perturbagens. Their biological
efficiency is evaluated in vitro in Homunculus bioreactor. Then preparation is stored as
described previously.

Individual DV is prepared from the leukoconcentrate of peripheral blood of the patient. The
lymphocytes are isolated, cultured with G-CSF and interleukin-2, conditioned by
tumor-specific antigens, TNF-α and PGE2 for 48 hours and loaded with recombinant proteins
identical to key tumor-specific antigens detected at proteomic testing of tumor cells (TCs).
Basic mechanism of DV immune effect is elaboration of tumor toxic lymphocytes by the
organism of the patient.

CTLs are obtained as described previously. The intervention is described in the appropriate
section.

Toxicity will be evaluated according to CTC-NCI criteria. Efficiency is assessed according
to the following criteria:

1. Complete effect - full disappearance of all tumor foci

2. Partial effect - reduction of tumor size and/or metastatic foci by no less than 50% and
no signs of new neoplasms

3. Stabilization - reduction of tumor foci size by less than 50% and no signs of new
neoplasms

4. Progress - growth of tumor foci during the therapy. In case of mosaic effect, when part
of foci progresses and part is stable or reducing, the therapy is continued but the
cases are analyzed outside the context "Response to the therapy"